Synthesis and aggregation behaviour of single-chain, 1,32-alkyl branched bis(phosphocholines): Effect of lateral chain length

Article abstract of DOI:10.1039/c8ob00424b

Three novel single-chain bis(phosphocholines) bearing two lateral alkyl chains of variable length next to the headgroup have been synthesized as model lipids for naturally occurring archaeal membrane lipids. The synthesis was realized using the Cu-catalyzed Grignard bis-coupling reaction of a primary bromide as a side part and a 1,ω-dibromide as a centre part. We could show that the aggregation behaviour of the resulting bolalipids strongly depends on the length of the lateral alkyl chain: the C3-branched bolalipid self-assembles into lamellar sheets, whereas the C6- and C9-analogues form nanofibres. The lamella-forming bolalipids could be used in the future to prepare stable and tailored liposomes for oral drug delivery.

Full text of DOI:10.1039/c8ob00424b

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Synthesis and aggregation behaviour of single-chain, 1,32-alkyl
branched bis(phosphocholines): effect of lateral chain length†
K. Gruhle,a S. Müller,a A. Meister,b and S. Drescher*a
Received 00th January 20xx,
Accepted 00th January 20xx
Three novel single-chain bis(phosphocholines) bearing two lateral alkyl chains of variable length next to the headgroup
have been synthesized as model lipids for natural occuring archaeal membrane lipids. The synthesis was realized using the
Cu-catalyzed Grignard bis-coupling reaction of a primary bromide as side part and a 1,ω-dibromide as centre part. We
could show that the aggregation behaviour of the resulting bolalipids strongly depends on the length of the lateral alkyl
chain: the C3-branched bolalipid self-assembles into lamellar sheets, whereas the C6- and C9-analogue forms nanofibres.
The lamella-forming bolalipids could be used in the future to prepare stable and tailored liposomes for oral drug delivery.
DOI: 10.1039/x0xx00000x
www.rsc.org/
bolalipids alone, which are sometimes called archaeosomes,11
or from bolalipid/phospholipid mixtures was already proved
for a wide range of natural and artificial bipolar lipids.12-18
Introduction
Bolaamphiphiles (or bolalipids) consist of a long hydrophobic
part, mainly one or two membrane-spanning (transmembrane)
alkyl chains, and two hydrophilic headgroups bound to both
ends of the alkyl spacer.1 These bipolar lipids can be found in
the membranes of certain species of archaea, e.g., thermoaci-
dophiles, where they are responsible for the great stability of
archaea against extreme living conditions, such as high tem-
peratures and/or low pH-values.2-4 This stability is reasoned in
the chemical structure of archaeal bolalipids that differs from
the structure of membrane lipids from eukaryotes and bacteria
in three important aspects:5,6 first, the glycerol backbone is in
the invers sn-2,3 configuration; second, the hydrophobic parts
(fatty alcohols) are bound via ether bond to the glycerol; and
third, the fatty alcohols contain isoprenoid-branched methyl
groups and/or a variable number of 1,3-bound cyclopentane
rings.2,3,5 The main alcohols found in archaea are the sn-2,3-
diphytanylglycerol diether (archaeol) and its dimer the sn-2,3-
dibiphytanyldiglycerol tetraether (caldarchaeol).
However, the main problem is to obtain such bolalipids in
considerable amounts for practical applications. Isolation from
natural sources is on the one hand a suitable method; but, this
method yields mixtures of lipids with respect to the composi-
tion of the hydrophobic part of the bolalipid. On the other
hand, the total synthesis of natural occurring archaeal mem-
brane lipids, which was shown by Kakinuma et al.,19-21 is not
applicable for the production of large batches. Consequently,
researchers in this field try to simplify the chemical structure
of bolalipids by maintaining their stabilizing properties.16,22-25
The simplest archaeal model lipid is, in our opinion, a single-
chain bolalipid consisting of two phosphocholine (PC) head-
groups connected by an unmodified C32 alkyl chain, the PC-
C32-PC (Figure 1, top).26
When put into water, PC-C32-PC self-assembles into long
nanofibres or micellar aggregates in dependence of the tem-
perature.27 The formation of vesicular structures (liposomes)
was not observed. This surprising aggregation behaviour is
caused by the chemical structure of this archaeal model lipid:
The extraordinary stability of archaeal membranes against
external stress, e.g. the presence of low pH-values, high tem-
peratures, or hydrolysing enzymes, motivates researchers to
use these membrane lipids in biotechnology,7 material scienc-
es,8 and pharmacy.9,10 Especially the possibility of bipolar lipids
to incorporate into bilayers composed of conventional mono-
polar phospholipids is very attractive, since this approach led
to stabilized liposomes, which can be used for drug delivery
purposes. The applicability of liposomes prepared from either
O
O
O
O
P
N
N
N
P
O
O
O
O
15
O
O
PC-C32-PC
R
O
P
O
P
O
O
N
a.Institute of Pharmacy, Biophysical Pharmacy, Martin Luther University (MLU)
Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale),
Germany. E-mail: simon.drescher@pharmazie.uni-halle.de
15
O
O
R
PC-C32(1,32C3)-PC R = nꢀC3H7
PC-C32(1,32C6)-PC R = nꢀC6H13
PC-C32(1,32C9)-PC R = nꢀC9H19
b.Institute of Biochemistry and Biotechnology, MLU Halle-Wittenberg, Kurt-Mothes-
Strasse 3, 06120 Halle (Saale), Germany.
†
Electronic Supplementary Information (ESI) available: DLS measurements; MS, 1H
Figure 1 Chemical structure of the archaeal model lipid PC-C32-PC and its 1,32-alkyl
and 13C NMR spectra. See DOI: 10.1039/x0xx00000x
branched derivatives investigated in this work.
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Due to the bulkiness of the PC headgroup in comparison the
small cross-sectional area of its single alkyl chain, the self-
assembly into lamellar structures is energetically unfavourable
and the aggregation into fibres is preferred.28 Within these
nanofibres, the PC-C32-PC molecules are twisted relative to
each other, which leads to a helical superstructure of the fi-
bres.29 However, PC-C32-PC cannot be used as stabilizer of
phospholipid bilayers (liposomes).30 The reason for that can be
found again in the chemical structure of the PC-C32-PC mole-
cule: if the bolalipid is inserted in a stretched, transmembrane
manner into the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) bilayer, void volume will be created in the hydrophobic
region between both lipid species, which cannot be filled by
either DPPC or PC-C32-PC. To circumvent these packing prob-
lems, the enlargement of the hydrophobic part of the bolalipid
by expanding the cross-sectional area of the alkyl chain—to fill
the void volume—is one possibility.
Method A: Grignard bisꢀcoupling
Mg
+
Y
O
A
X
Br
B
Br
Y
O
A
B
A
O Y
[Cu]
R
R
R
R = H, alkyl side chain
X = Br, tosyl
Y = THP, trityl
A
B
C
alkyl chain
of variable length
Method B: Grignard monoꢀcoupling
Mg
+
Y
O
A
X
Br
C Othp
Y
O
A
C
Othp
[Cu]
R
R
1. thp
2. Mg / [Cu]
Br
R = H, alkyl side chain
X = Br, tosyl
Y = trityl, benzyl
Y
O
A
C
C
A
O Y
R
R
Figure
2 Schematic overview of Grignard coupling reactions. Top: Grignard bis-
coupling of a side part A and a centre part B in a 2:1 ratio; bottom: Grignard mono-
coupling consisting of two steps, a Grignard coupling of A + C and a dimerization of the
A–C product. For both methods, either part A or part B/C can be transformed in the
corresponding Grignard reagent.
In recent years, we systematically variated the alkyl chain
structure of single-chain bolalipids through the insertion of
hetero atoms,31,32 acetylene33 or diacetylene34 groups, phenyl-
as well as biphenyl rings,35-38 and also methyl moieties.33 We
could show that perturbations within the alkyl chain led to a
decrease of the nanofibre stability and, in some cases, elon-
gated micelles are formed instead. However, in mixing experi-
ments of these modified bolalipids with classical phospholipids
we could demonstrate that if a miscibility of both lipid species
was observed, the liposomal structure is destroyed and small
disk-like aggregates are found instead.38,39 We think that small
alterations in the bolalipid alkyl chain such as two methyl
groups39 are not sufficient to fill the void volume. Possibly, the
insertion of longer alkyl (side)chains into the transmembrane
C32 chain of the PC-C32-PC could result in a better miscibility
of bolalipids with phospholipid by maintaining the vesicular
structure.
double Wittig reaction with bis(phosphorylides) and ω-
functionalized aldehydes.40,41 In recent years, we established
and elaborated the Grignard reaction to build up long-chain
1,ω-diols. This C-C-coupling reaction, which is catalyzed by
Li2CuCl4,42 can be designed as a mono-coupling43 or bis-
coupling26,44 reaction (Figure 2).
The Grignard bis-coupling involves two simultaneously per-
formed C-C-coupling reactions using a side part
A and a centre
part B in a molar ration of 2:1 (see Figure 2, method A). This
reaction results in the formation of a A-B-A product. The main
drawback of this approach is that due to a transmetallation
reaction, firstly described by Tamura and Kochi,42 an A-A prod-
uct is formed (which we term homo-coupling product). In
some cases, this by-product cannot be separated from the
main product A-B-A 36,44
With the use of a Grignard mono-coupling of two different
parts and , one can avoid this problem (see Figure 2, meth-
.
In this paper, we present the synthesis of three novel alkyl-
branched single-chain bis(phosphocholines) PC-C32(1,32Cn)-
A
C
PC. These bolalipids carry two side chains at the
α,α’(1,32)-
od B). Of course, this coupling reaction also leads to homo-
coupling by-products A-A or C-C, but, due to a sufficient differ-
ence in chemical structures (polarity), both by-products can be
fully separated from the main product A-C using column
chromatography.36 Finally, A-C is dimerized to the final product
A-C-C-A using a second Grignard homo-coupling reaction. The
increased effort (more synthetic steps) is sometimes necessary
to avoid non-separable by-products.
position of the long C32 chain with different chain lengths of n
= 3, 6, or 9 carbon atoms (Figure 1, bottom). We will also dis-
cuss scopes and limits of the Grignard coupling reaction that is
used for the preparation of the alkyl-branched diols. In the
second part of the paper, we present first investigations of the
lyotropic behaviour of these novel bolalipids in aqueous sus-
pension by differential scanning calorimetry (DSC), turbidity
measurements, and transmission electron microscopy (TEM).
In a first attempt, we used the Grignard bis-coupling reaction
(method A). Since the total bolalipid chain length is set to 32 C-
atoms and besides, commercially available
δ-lactones (C5
Results and discussion
building block) are used as starting material for the prepara-
tion of the side parts, a centre part of 22 C-atoms has to be
synthesized at first. The 1,22-dibromodocosane (3a) was pre-
pared from the tetrahydropyranyl(THP)-blocked 11-
Synthetic methods
The preparation of single-chain bolalipids depends on an effec-
tive synthetic access to the corresponding diols. For the syn-
thesis of 1,ω-diols with 22 or more carbon atoms, several
procedures are known: (i) bis-acylation of cyclic enamines with
dicarboxylic acid dichlorides, (ii) hydrolysis of enamino ketones
and subsequent ring opening, (iii) Wolff-Kishner reduction of
bis(oxoacids) and subsequent reduction with LiAlH4, or (iv)
bromoundecanol (
1
).44 After the Li2CuCl4-catalyzed Grignard
the resulting bis-THP-ether was con-
homo-coupling of 1 45
,
2
verted into the favoured 3a using triphenylphosphoranediyl
dibromide and a procedure described by Schwarz et al.
(Scheme 1).46
2 | Org. Biomol. Chem., 2018, 00, 1-3
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first entry (see Table 1 on the next page) using 3a failed. It was
not possible to convert the dibromide into the organometallic
compound, presumably due to the poor solubility of 3a in
tetrahydrofuran (THF). It was also not possible to convert the
O
O
Br
O
O
i
O
O
5
10
1
2
secondary bromide
9 into the Grignard reagent (entry 2),
ii
which might be due to steric hindrance. Also a prolonged reac-
tion time or the addition of iodine or 1,2-dibromoethane to
activate the magnesium did not work. Yang et al. suggested
the addition of N,N,N’,N’-tetramethylethane-1,2-diamine
(TMEDA), which act as a chelator and promote the formation
of secondary organometallic compounds.50 But, this attempt
was not crowned with success, too (entry 3).
Br
Br
n
(3a n = 10
(3b n = 3)
Scheme 1 Reagents and conditions for the preparation of the long-chain dibromide
3a: (i) Mg, 1, THF, 50 °C; then 1, Li2CuCl4, THF, 0 °C; (ii) PPh3, Br2, CH2Cl2, r.t.; 1,8-
dibromooctane (3b) is commercially available.
Since Grignard coupling reactions can also be carried out us-
ing pseudohalides such as mesylates or tosylates as coupling
reagents,50-52 we wanted to use secondary tosylates instead of
bromides. Therefore, we synthesized two different tosylates
As mentioned above, commercially available racemic
nonalactone ( ) was used as starting material for the prepara-
tion of the side part (Scheme 2). Compound was converted
into the isopropyl (5RS)-5-hydroxynonanoate ( ) by a base-
δ-
4
starting from either racemic δ-lactones or hydroxy fatty acids
4
(Scheme 3). Besides the base-induced lactone ring opening
described above and the acid-catalyzed transesterification
published elsewhere,30 lactones 10 could also be cleaved by
reduction using LiAlH4 in diethyl ether. This reduction led to
the diols (5RS)-octane-1,5-diol (12a) and (5RS)-tetradecane-
1,5-diol (12b) in good yields including a primary as well as a
secondary alcohol function. One can also start from the com-
mercially available racemic 12-hydroxy-stearic acid (11). After
the reduction of the carboxylic group, the (12RS)-octadecane-
1,12-diol (12c) was obtained (Scheme 3). The next step is the
selective protection of one hydroxy moiety of the diols 12. We
decided to use the trityl group to selectively block the primary
alcohol moiety. Subsequently, the secondary hydroxy group
was tosylated using tosyl chloride (TsCl). Both reactions can be
performed as a one-pot synthesis using a chloroform/pyridine
5
induced lactone ring opening followed by a esterification using
2-bromopropane.47 The best yields could be obtained using
N,N-dimethylformamide (DMF) and a sterically hindered alco-
hol (tert-butanol) as solvents. One can also apply an acid-
catalyzed ring opening reaction by heating
4 in MeOH with
catalytic amounts of p-toluenesulfonic acid.44 However, this
acidic transesterification led to a high amount of oligomer
products. The next step was the conversion of the hydroxy
group into a bromide. Here, we used again the procedure
described by Schwarz et al.46 and transferred the hydroxy ester
5
into the corresponding THP-ether 6 48
.
After bromination, the
isopropyl (5RS)-5-bromononanoate (
7) was obtained. The last
step was the reduction of the ester function using LiAlH4 in
diethyl ether. This reaction has to be performed at −15 °C to
prevent a possible reductive elimination of the secondary
bromide. Finally, the primary alcohol function of the resulting
R
O
O
OH
O
(5RS)-5-bromononan-1-ol (
8
) was blocked via THP-ether yield-
).48
R
OH
ing the 2-{[(5RS)-5-bromononyl]oxy}tetrahydro-2H-pyran (
9
8
10a R = nꢀC3H7
10b R = nꢀC9H19
i
i
11 R = nꢀC6H13
The first Grignard reactions were carried out in a bis-coupling
manner (Figure 2, method A) using the dibromide 3a as centre
OH
part and the secondary bromide 9 as side part according to the
literature.49 Now we have two options: We can transform
R
OH
m
12a m = 1, R = nꢀC3H7
12b m = 1, R = nꢀC9H19
12c m = 8, R = nꢀC6H13
either 3a or
9 into the corresponding Grignard reagent. The
ii
R
O
iii
secondary tosylates
i
Otosyl
OH
O
O
O
iii
R
Otrityl
R
Br
m
4
5 R = OH
m
ii
13b m = 1, R = nꢀC9H19
13c m = 8, R = nꢀC6H13
14a m = 1, R = nꢀC3H7
14b m = 1, R = nꢀC9H19
14c m = 8, R = nꢀC6H13
6
7
R = Othp
R = Br
Br
iv
OH
iv
primary bromides
Othp
8
ii
secondary bromide
R
Br
m
Br
15a m = 1, R = nꢀC3H7
15b m = 1, R = nꢀC9H19
15c m = 8, R = nꢀC6H13
O
O
9
Scheme 3 Reagents and conditions for the preparation of secondary tosylates 13 and
primary bromides 15: (i) LiAlH4, Et2O, r.t.; (ii) TrCl, pyridine, CHCl3, r.t.; then TsCl,
pyridine, CHCl3, r.t.; (iii) PPh3, CBr4, CH2Cl2, 10 °C; (iv) DHP, CH2Cl2, PPTS, r.t.
Scheme 2 Reagents and conditions for the preparation of the secondary bromide 9: (i)
KOH, MeOH, r.t.; then 2-bromopropane, DMF, tert-butanol, reflux; (ii) DHP, CH2Cl2,
PPTS, r.t.; (iii) PPh3, Br2, CH2Cl2, r.t.; (iv) LiAlH4, Et2O, −15 °C.
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Table 1 Reagents and conditions for the Grignard coupling reactions performed in this work.a
Entry
Method
(see
Educt 1
(Grignard
reagent)
3a
Yieldb (%)
of Grignard
reagent
Educt 2
(coupling
reagent)
9
Ratio
Cat.
Additive
Temp. of
coupling
reactiond
Product
Yielde (%)
Ed1 : Ed2
(10 mol%)c
(20 mol%)
Figure 2)
g
1
2
A
A
A
B
B
B
A
A
A
A
A
A
< 5
< 5
< 5
73
1 : 2
Li2CuCl4
Li2CuCl4
Li2CuCl4
Li2CuCl4
Li2CuCl4
CuI, LiOMe
Li2CuCl4
CuI, LiOMe
Li2CuCl4
Li2CuCl4
Li2CuCl4
Li2CuCl4
–
0 °C
0 °C
–
–
–
(1.78 mmol)
9
(3.55 mmol)
3a
g
2.1 : 1
2.1 : 1
1.1 : 1
1.3 : 1
2 : 1
–
–
(3.55 mmol)
9
(1.69 mmol)
3a
g
3
TMEDAf
0 °C
–
–
(2.86 mmol)
1
(1.36 mmol)
13b
h
4
–
0 °C
–
–
(4.92 mmol)
1
(4.36 mmol)
13b
h
5
75
–
0 °C → r.t.
0 °C → r.t.
0 °C
–
–
(4.56 mmol)
1
(3.51 mmol)
13b
h
6
76
TMEDA
–
–
(9.19 mmol)
3b
(4.60 mmol)
13c
h
7
70
1 : 2.2
1 : 2.2
2.9 : 1
1 : 2.1
1 : 2.5
2 : 1
–
–
–
(1.98 mmol)
3b
(4.36 mmol)
13c
h
8
75
TMEDA
0 °C → r.t.
0 °C → r.t.
0 °C
–
–
(1.33 mmol)
15a
(2.93 mmol)
3a
9
> 98
> 98
> 98
89
–
–
–
–
16a
16b
16b
16c
44
14
59
13
(26.77 mmol)
3b
(9.16 mmol)
15c
10
11
12
(3.93 mmol)
3b
(8.71 mmol)
15c
0 °C → r.t.
0 °C
(7.17 mmol)
15b
(17.69 mmol)
3a
(11.98 mmol)
(5.96 mmol)
a All reactions were performed in THF. b Yields determined by weighing the Mg left after Grignard formation. c 10 mol% of Cu-catalyst with respect to the molar amount
d
of Grignard reactions. The formation of the Grignard (MgBr) reagent was done at 50 °C, whereas the subsequently performed coupling reaction was done at the
temperature mentioned in the table (r.t. = room temperature). e Isolated yields after chromatography. f N,N,N’,N’-tetramethylethane-1,2-diamine. g Reaction was not
successful because no Grignard reagent was formed. h Reaction was not successful because no coupling reaction was observed.
mixture as solvent.53 It has to be mentioned that in some cases and 8, Table 1). Again, no product formation was observed in
a
β
-elimination of the secondary tosyl group was found, which both trials. That means Grignard coupling reactions using long-
resulted in lower yields. The obtained 1-[(4RS)-4-(trityloxy)- chain secondary bromides or tosylates are not feasible for the
butyl]decyl 4-methylbenzenesulfonate 13b and 1-hexyl- synthesis of long-chain alkyl-branched diols.
(
)
[(12RS)-12-(trityloxy)dodecyl] 4-methylbenzenesulfonate (13c
)
But, we did not get discouraged by these backlashes. Since
we have very good experiences with Grignard coupling reac-
can be used for the subsequent Grignard reactions.
For the next set of Grignard reactions, we used the mono- tions of two primary bromides,36,43,44 we synthesized a set of
coupling methodology (Figure 2, method B). As first part, we such primary bromides including a blocked secondary alcohol
used the THP-blocked 11-bromoundecanol44
(1), which was moiety (compounds 15, Scheme 3). The preparation started
transferred into the corresponding Grignard reagent. The from diols 12, which we already had on hand. Now, the crucial
second part was the secondary tosylate 13b. The reaction of step is the selective bromination of the primary hydroxy group.
both components should lead to a C16 building block with a De Luca et al. suggested
THP- and trityl-blocked hydroxy moiety. After
trichloro[1,3,5]triazine and DMF54,55 to obtain a primary chlo-
THP
bromine exchange,46 this product could be dimerized ride, which could subsequently be substituted by bromine
a
reaction with 2,4,6-
a
a
→
to the alkyl-branched C32 diol. Unfortunately, all attempts using LiBr. Unfortunately, the first step was not selective and
(entries 4–6, Table 1) were not successful. Neither an in- both primary and secondary chlorides were obtained. Another
creased temperature of the coupling reaction (entry 5) nor a possibility is the direct bromination using the Appel reaction.56
change in the Cu-catalyst50 (entry 6) resulted in a notable For this purpose, the diols 12 were get into reaction with tri-
product formation. We then went back to the Grignard bis- phenylphosphane and tetrabromomethane in methylene chlo-
coupling (Figure 2, method A) and used the short-chain 1,8- ride. The yields of this nucleophilic substitution reaction could
dibromooctane (3b) as centre part and the secondary tosylate be increased from 40% to about 70% by changing the reaction
13c as side part. After the formation of the Grignard reagent temperature from 0 °C to 10 °C. The selectivity of this reaction
1
from the dibromide 3b, we tested the Grignard coupling with was proved by H NMR experiments. In the last step, the sec-
13c using two different catalysts and temperatures (entries 7 ondary hydroxy group of the obtained racemic bromoalcohols
4 | Org. Biomol. Chem., 2018, 00, 1-3
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14 was protected as THP-ether.48 The resulting racemic THP- form/triethylamine (TEA) mixture. The subsequently per-
blocked bromoalcohols 15 were obtained in very good yields formed quarternisation with trimethylamine in a chloro-
(>95%) and can be used for the Grignard reaction.
form/acetonitrile/ethanol
mixture
provided
the
For the last set of Grignard reactions (entries 9–12, Table 1) bis(phosphocholines) PC-C32(1,32n)-PC in 11–33% yield after
we used the bis-coupling methodology (Figure 2, method A) purification and with respect to the diols 17 (Scheme 4). The
and the different THP-blocked bromoalcohols 15 as side parts relatively low yields are due to the secondary alcohol function.
and the 1,ω-dibromides
3 as centre part. Since the formation It is conceivable the sterically hindrance reduces the yields
of the Grignard reagent from 1,22-dibromodocosane (3a) was during the phosphorylation reaction. For comparison, the
not possible (see entry 1, Table 1, and description above), we yields for the phosphorylation/quarternisation reaction of
used this compound as coupling partner (entries, 9 and 12). By unmodified long-chain 1,ω-diols are between 54 and 65%.
contrast, we prepared the Grignard reagent from the 1,8-
Temperature-dependent aggregation behaviour
dibromooctane (3b) in entry 10 and 11. After the Grignard
coupling reaction using Li2CuCl4 as catalyst, we obtained the The lyotropic phase behaviour of the novel, single-chain alkyl-
corresponding alkyl-branched bis-THP-ethers 16 in moderate branched bolalipids PC-C32(1,32n)-PC was characterised by
yields (Scheme 4). In contrast to previous work,36,44 the yield DSC, turbidity measurements, and TEM. The results were fur-
could be significantly increased when the temperature of the ther compared to the unmodified PC-C32-PC. As already men-
coupling reaction is increased from 0 °C (entries 10, 12; 13– tioned in the introduction, PC-C32-PC self-assembles in aque-
14%) to room temperature (entries 9, 11; 44–59%; Table 1). ous suspension into a dense network of long flexible, helical
After cleavage of both THP protecting groups, the correspond- nanofibres. This leads to the formation of a clear and transpar-
ing 1,32-alkyl-branched diols 17 were obtained in good yields ent hydrogel.26 With increasing temperature, a transformation
of about 88 %.
of the nanofibres into small micelles can be observed and the
Finally, the 1,32-alkyl-branched bis(phosphocholines) PC- gel character is lost. This reversible gel/sol transformation is
C32(1,32n)-PC were prepared from the corresponding diols 17 accompanied by an endothermic and cooperative transition at
by the method described by Eibl et al.57 using
β
-
Tm1 = 48 °C, which can be followed by DSC.27 At higher tem-
bromoethylphosphoric acid dichloride58 as phosphorylating peratures (Tm2 ≈ 73 °C), a second broad transition between
reagent. The phosphorylation step has to be performed at two different types of micelles is observed (Figure 3, grey solid
higher temperatures for a prolonged period of time (about 24 line).27
h at 50 °C and up to 3 days at room temperature) in order to
DSC and Turbidity. When put the new alkyl-branched bo-
ensure a complete dissolution of the diols 17 in the chloro- lalipids in water, a different behaviour can be observed. The
C3-branched PC-C32(1,32C3)-PC forms a turbid suspension,
Othp
which becomes completely clear when heated above 80 °C and
changes into a turbid suspension again when cooled. In con-
Br
+
Br
R
Br
m
n
3a n = 10
3b n = 3
15a m = 1, R = nꢀC3H7
15b m = 1, R = nꢀC9H19
15c m = 8, R = nꢀC6H13
70
60
i or ii
R
16a R = nꢀC3H7
(15a + 3a, i)
50
PC-C32-PC
O
O
16b R = nꢀC6H13
(3b + 15c, ii)
O
O
15
40
R
R
R
16c R = nꢀC9H19
(15b + 3a, i)
PC-C32(1,32C6)-PC
iii
R
30
PC-C32(1,32C3)-PC
20
10
HO
17a R = nꢀC3H7
17b R = nꢀC6H13
17c R = nꢀC9H19
OH
15
iv
R
PC-C32(1,32C9)-PC
0
O
P
O
O
O
N
ꢀ10
N
P
O
O
heating
15
O
O
cooling
ꢀ20
PC-C32(1,32C3)-PC R = nꢀC3H7
PC-C32(1,32C6)-PC
R = nꢀC6H13
PC-C32(1,32C9)-PC R = nꢀC9H19
10 20 30 40 50 60 70 80
Temperature / °C
Scheme 4 Reagents and conditions for Grignard coupling reactions and final steps to
the bolalipids: (i) Mg, 15a or 15b, THF, 50 °C; then 3a, Li2CuCl4, THF, 0 °C or r.t.; (ii) Mg,
3b, THF, 50 °C; then 15c, Li2CuCl4, THF, 0 °C or r.t.; (iii) MeOH, PPTS, reflux; (iv)
Cl2P(O)O(CH2)2Br, TEA, CHCl3, 50 °C; then THF, H2O, r.t.; then CHCl3, acetonitrile, EtOH,
N(CH3)3, 40 °C.
Figure 3 DSC heating (solid lines) and cooling (dotted lines) curves of aqueous suspen-
sions of bolalipids PC-C32(1,32Cn)-PC (c = 1 mg mL−1, heating rate 60 K h−1). The DSC
curve of PC-C32-PC is shown for comparison. The curves are shifted vertically for
clarity.
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Organic and Biomolecular Chemistry
trast, the C6-branched PC-C32(1,32C6)-PC forms a transparent
and gel-like suspension, which stays clear during heating. Final-
ly, the C9-branched PC-C32(1,32C9)-PC also forms a transpar-
ent suspension, which is less gel-like compared to the C6-
analogue. But, with increasing temperature, the PC-
C32(1,32C9)-PC suspension turns into a turbid one and small
particles start to precipitate.
B
80
18
A
35
30
25
20
15
10
5
C
60
50
40
30
20
10
0
16
14
12
10
8
80
60
40
20
0
80
60
40
20
0
60
40
20
0
6
4
2
0
0
20 30 40 50 60 70 80
10 20 30 40 50
10
20
30
40
The DSC heating curves of the alkyl-branched bolalipids in
water (c = 1 mg mL−1) are depicted in Figure 3. The PC-
C32(1,32C3)-PC, with two n-propyl side chains, shows a coop-
erative transition at Tm = 63.2 °C including a small high-
temperature shoulder (red solid line in Figure 3). A further
elongation of the lateral side chain leads to a pronounced
decrease in the transition temperature: PC-C32(1,32C6)-PC
shows a cooperative, endotherm transition at Tm = 20.7 °C
(green solid line in Figure 3). In addition, a stepwise decrease
in the heat capacity is observed between 30–42 °C. This de-
crease is conceivably related to a metastable state that is
reached after Tm. This kinetically stable state then transforms
within the timescale of the DSC experiment into a thermody-
namically ones, which is accompanied by an exothermic heat
effect showing up as decrease in the Cp value. The bolalipid
including the longest side chain, the PC-C32(1,32C9)-PC, shows
a cooperative transition at Tm = 20.8 °C and a very small transi-
tion at T = 25.8 °C (blue solid line in Figure 3). Above 70 °C, the
DSC signal becomes noisy, which might be due to precipitation
of lipid within the DSC cell. The drastic decrease in Tm of the
C6- and the C9-analogue, when compared to PC-C32(1,32C3)-
PC as well as PC-C32-PC, is obviously due to the longer alkyl
side chains. A comparable effect—although not as pronounced
as in our case—was shown for alkyl-branched, monopolar
phosphatidylcholines.59 Here, the C3-branched 1,2-di(2C3-
16:0)PC depicts a main transition temperature at Tm = 29.4 °C,
whereas the C6-analogue 1,2-di(2C6-16:0)PC reveals Tm = 3.0
°C.
Temperature / °C
Temperature / °C
Temperature / °C
Figure
4
Temperature-dependent transmission measurements (red squares) of
1mg mL−1): (A) PC-C32(1,32C3)-PC, (B) PC-
aqueous bolalipid suspensions (c
=
C32(1,32C6)-PC, (C) PC-C32(1,32C9)-PC. The DSC heating curves (black solid line) of
the corresponding bolalipid suspensions (c = 1 mg mL−1; 60 K h−1) are shown for
comparison.
ture-dependent turbidity measurements. The corresponding
transmission versus temperature graphs are shown in Figure 4.
For the PC-C32(1,32C3)-PC, we found an increase in the
transmission from 60% at T = 20 °C to 90% at T = 70 °C (Figure
4A). This increase is most pronounced at Tm. The PC-
C32(1,32C6)-PC stays more or less clear and transparent over
the whole temperature range investigated with transmission
values of about 90% (Figure 4B). Lastly, the PC-C32(1,32C9)-PC
starts with a virtually clear suspension and a transmission
value of about 75% at T = 10 °C. During heating, the transmis-
sion value drops down to 20% at temperatures between 20–24
°C, which corresponds to Tm. Above 25 °C, the suspension
(supernatant) becomes clear again (transmission of 90% at T =
35 °C), which is due to precipitation of the lipid substance
(Figure 4C).
TEM. To get an idea about the shape of aggregates formed in
water at different temperatures, i.e. below and above Tm, and
in order to find an explanation for the different turbidity phe-
nomena, TEM images were recorded from negatively stained
samples (c = 0.05 mg mL−1, Figure 5).
The PC-C32(1,32C3)-PC with the shortest alkyl side chain (C3)
self-assembles at room temperature (T ≈ 22 °C), below Tm, into
sheet-like aggregates of different size and irregular shape
(Figure 5A, B). Closed lipid vesicles were not found. The inser-
tion of two short (C3) alkyl side chains obviously prevents the
formation of nanofibres as found for the unmodified PC-C32-
The corresponding cooling curves of the alkyl-branched bo-
lalipids are shown in Figure 3 as dotted lines. The PC-
C32(1,32C3)-PC shows a very broad transition between T = 65–
50 °C and the main transition at Tm = 33.4 °C, indicating a pro-
nounced hysteresis of about 30 K (Figure 3, red dotted line).
The PC-C32(1,32C6)-PC depicts a main transition at Tm = 18.8
°C, which equals a very small hysteresis of about 2 K (Figure 3,
green dotted line). Finally, the PC-C32(1,32C9)-PC shows a
transition at Tm = 14.9 °C, indication a moderate hysteresis of
PC 26,27
Due to the high Tm-value of PC-C32(1,32C3)-PC, we
.
were not able to get an EM image of the aggregates formed
above Tm. Since the suspension becomes clear at temperatures
above Tm (see Figure 4A), it is conceivable that the sheet-like
aggregates transform into small micelles or nanofibres at these
high temperatures. DLS measurements at T = 80 °C (see Figure
S1, ESI†) reveal the presence of very small particles with a
diameter between 10–14 nm, which can be attributed to mi-
cellar structures. However, also larger aggregates are present
in the sample: a second population with diameters around 200
nm and a third population with very large particle sizes > 1 µm.
Both could be attributed to fibrous aggregates, since the sam-
ple is virtually clear.
ꢀT = 6 K (Figure 3, blue dotted line). It might be conceivable
that the short C3 side chain in the PC-C32(1,32C3)-PC leads to
higher perturbation in the aggregate structure than the C6 and
C9 ones, which results in a more hindered reorganisation and,
hence, a larger hysteresis. Interestingly, the DSC cooling curve
of PC-C32-PC shows only very broad and no sharp and cooper-
ative transitions (Figure 3, grey dotted line). Probably, the
additional alkyl side chains in the novel bolalipids trigger a
faster (within the timescale of the DSC measurement) reorgan-
isation from the high-temperature to low-temperature aggre-
gate structures.
The situation changes when the lateral chain is elongated.
The C6-analogue PC-C32(1,32C6)-PC shows the formation of a
dense network of nanofibres (Figure 5C), when the sample is
prepared below Tm. These fibres appear as single fibre strands
The variable and changing turbidity of the different bolalipid
suspensions described above can also be followed by tempera-
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5E, F; samples prepared at T = 35 °C), these nanofibres seems
to form circular structures. This aggregations behaviour was
observed in several independently prepared samples, thus
preparation artefacts can be ruled out. Similar structures—
although not as pronounced as in this case—have been ob-
served for an asymmetrical single-chain bolalipid named
DMAPPC-C32-POH in acetate buffer at pH 5.60 The reason for
this transformation as well as the inner structure of these
circular aggregates are not understood up to now and are part
of ongoing research.
A
B
200 nm
200 nm
The bolalipid bearing the longest (C9) lateral alkyl chains, PC-
C32(1,32C9)-PC, self-assembles again into long and flexible
nanofibres below Tm (Figure 5G), which lead to a gelation of
the bolalipid suspension. These nanofibres are comparable to
the fibres found for PC-C32(1,32C6)-PC and PC-C32-PC. Upon
heating, the fibres transform into lamellar aggregates of ir-
regular shape (Figure 5H; sample prepared at T = 35 °C), which
is accompanied by an increase in turbidity (see Figure 4C) and
a loss of the gel properties.
C
D
500 nm
100 nm
Geometrical considerations. By comparing the three alkyl-
branched bolalipids PC-C32(1,32Cn)-PC investigated in this
work, one can conclude that all of them show a different ag-
gregation behaviour in dependence of temperature—
summarized in Figure 6. The bolalipids only differ in the length
of the lateral alkyl chain. First temperature-dependent infra-
red(IR)-spectroscopic measurements of aqueous bolalipid
suspensions reveal that the transition found in the DSC is ac-
companied with an increased number of gauche conformers
within the alkyl chains of the bolalipids (data not shown).
Hence at Tm, the bolalipids undergo a transition from gel phase
into a liquid-crystalline phase. This fluidization is connected
with a shortening of the alkyl chains. One can now speculate
that the interaction of the two factors, one the one hand the
length of the lateral alkyl chain and on the other hand the
reduction of the overall chain length upon heating, determine
the shape of aggregates found in aqueous suspension. Howev-
er, further investigations are mandatory to prove this assump-
tion.
E
F
500 nm
100 nm
G
H
200 nm
100 nm
Conclusions
Figure 5 TEM images of aqueous suspensions of PC-C32(1,32Cn)-PC (c = 0.05 mg mL−1
)
prepared at different temperatures: (A, B) PC-C32(1,32C3)-PC, (C–F) PC-C32(1,32C6)-
PC, (G, H) PC-C32(1,32C9)-PC; (A, B) 22 °C, (C, D, G) 4–7 °C, (E, F, H) 35 °C. Samples
were stained with uranyl acetate before drying.
In this work, we have developed a synthetic strategy for the
preparation of single-chain alkyl-branched bis(phospho-
cholines). For the synthesis of 1,ω-diols bearing lateral alkyl
or as bundles of several fibres. A closer inspection reveals that
a single fibre has a diameter of 5–6 nm (Figure 5D), which
roughly corresponds to the length of a bolalipid molecule. The
entanglement of these fibres leads to a gelation of the bo-
lalipid suspension. A comparable behaviour was previously
found for the single-chain unmodified bis(phosphocholine) PC-
lateral
chain length
Aggregation behaviour PC-C32(1,32Cn)-PC
turbid
sheet-like structures
clear
micelles/fibres
C3
C6
C9
clear clear
nanofibres circular structures of nanofibres
C32-PC 26,27
That means the insertion of two lateral C6 alkyl
.
clear turbid
nanofibres sheet-like structures
chains at the 1,32-position of the long alkyl chain does not
alter the aggregation behaviour and the formation of fibres is
still possible—in contrast to PC-C32(1,32C3)-PC. A tempera-
ture increase above Tm leads to a change in the aggregation
behaviour: although fibrillar aggregates are still present (Figure
10
20
30
40
Temperature / °C
50
60
70
80
Figure 6 Comparison of the aggregation behaviour of aqueous suspension of the alkyl-
branched PC-C32(1,32Cn)-PC (c = 1 mg mL−1).
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chains of variable length, we finally used a Li2CuCl4-catalyzed products was carried out by recrystallization or by column
Grignard bis-coupling reaction of primary bromides as side chromatography using a middle pressure liquid chromatog-
parts—including a THP-blocked secondary alcohol—and 1,ω- raphy (MPLC) system by Büchi (Essen, Germany) and silica gel
dibromides as centre part. This coupling reaction should be 60 (Merck, 0.063–0.200 mm). Melting points were determined
carried out at ambient temperatures instead of 0 °C to in- with Boetius apparatus. Elemental analyses were carried out
crease the yield. The corresponding primary bromides can be on an Elemental Vario EL (Elementar Analysensysteme GmbH,
1
13
prepared in three steps from inexpensive alkyl-branched
δ-
Langenselbold, Germany). H and C NMR spectra were rec-
lactones or hydroxy fatty acids. Other attempts using second- orded on an Agilent Technologies 400 MHz VNMRS spectrome-
ary bromides or tosylates for the Grignard coupling reaction as ter (400/100 MHz) or an Agilent Technologies 500 MHz DD2
well as the use of additives such as TMEDA did not work in our spectrometer (500/125 MHz) with the use of CDCl3 or CD3OD
case. For the final insertion of the PC headgroups, robust and as internal standard. Chemical shifts (δ) are reported in parts
established procedures were used. The synthetic strategy per million (ppm). The coupling constants (J) are reported in
presented herein is also applicable for the preparation of bo- Hz. Mass spectrometric data were obtained with a Finnigan
lalipids bearing other lengths of alkyl side chains.
LCQ-Classic (ESI-MS; Thermo Separation Products, San José,
The lyotropic phase behaviour of the novel single-chain alkyl- USA) or an expressionS compact mass spectrometer (APCI-MS;
branched bolalipids PC-C32(1,32Cn)-PC (n = 3, 6, or 9) was Advion Inc., Ithaca, USA). High-resolution mass spectra (HRMS)
investigated using DSC and TEM. We could show that the were recorded on a Thermo Fisher Scientific LTQ-Orbitrap
length of the lateral alkyl chain has a great impact on the mass spectrometer (Waltham, USA) with static nano-
structure of aggregates formed in aqueous suspension as well electrospray ionization. Analytical HPLC was performed on a
as their stability upon heating. Whereas PC-C32(1,32C3)-PC Lichrospher Si 60–5 µm–125
× 4 mm column, using a PU 980
self-assembles into sheet-like structures at ambient tempera- Intelligent HPLC-pump and a LG-1580-02 Ternary Gradient Unit
ture, which are stable up to Tm = 63 °C, the C6- and the C9- (Jasco) and a SEDEX 55 ELS detector (SEDERE, France). The
analogue form a network of nanofibres below Tm leading to a following eluents were used: solvent A = CHCl3/MeOH/H2O
gelation of the bolalipid suspension. With increasing tempera- (42/42/16, v/v/v), B = CHCl3/MeOH/H2O (45/45/10, v/v/v);
ture, the long fibres of PC-C32(1,32C6)-PC transform into circu- gradient: 14 min 0
→ 100% A, 9 min 100 % A, 6 min 100 → 0%
lar structures at Tm, whereas the fibrous aggregates of PC- A, 5 min 0% A; flow 1 mL min−1.
C32(1,32C9)-PC convert into lamellar sheet-like structures. It is
Synthesis of the long-chain 1,ω-dibromide 3a
conceivable that the nanofibres of PC-C32(1,32Cn)-PC are
composed of bola molecules that are arranged side by side. By 2,2’-[Docosane-1,22-diylbis(oxy)]bis(tetrahydro-2H-pyran)
contrast, the lamellar aggregates of PC-C32(1,32Cn)-PC are (2).45 Magnesium turnings (1.6 g, 65.8 mmol) were placed in a
probably composed of a bolalipid monolayer. The detailed 100 mL round-bottomed flask under argon atmosphere. A
molecular arrangement of the bolalipid molecules within the solution
1 (14.56 g, 43.5 mmol) in dry THF (20 mL) was added
different aggregates as well as the reason for the transfor- dropwise. After the exothermic reaction had subsided, the
mation between different aggregate types are not understood mixture was stirred at 55 °C for 3 h. Afterwards, the excess
up to now and are part of ongoing research.
magnesium was removed under argon atmosphere and the
Our investigations show that the aggregation behaviour of Grignard solution was cooled to 0 °C. A freshly prepared solu-
single-chain alkyl-branched bolalipids can be tuned by varying tion of dilithium tetrachlorocuprate(II) (0.1 M in THF, 7.0 mL)
the length of the lateral alkyl chain. This fact is of particular was added with stirring. After a solution of
1 (11.93 g, 35.6
interest since lamella-forming bolalipids, which can also be mmol) in dry THF (70 mL) was added in one portion, stirring
synthesized in larger scale, could be used in the future to stabi- was continued for 20 h at 0 °C. For work-up, diethyl ether (150
lise phospholipid liposomes applicable for oral drug delivery. mL) was added and the resulting mixture was poured into an
Investigations regarding the mixing behaviour of our novel ice-cold saturated solution of ammonium chloride (150 mL).
bolalipids with classical phospholipids are currently under way. The organic layer was separated and the aqueous phase was
extracted several times with diethyl ether. The combined ethe-
real phases were washed with brine, dried over sodium sul-
Experimental section
phate and concentrated to dryness under reduced pressure.
The crude product was purified by column chromatography
General
using heptane/triethylamine/diethyl ether (98/0.5/1.5, v/v/v)
Apart from 12-hydroxyoctadecanoic acid (11; Fluorochem Ldt,
1
as eluent yielding
MHz, CDCl3, 27 °C)
H, 2 CHHCHO), 1.78–1.88 (m, 2 H, 2
9.6, 6.7 Hz, 2 H, 2 CHHOthp), 3.46–3.54 (m, 2 H, 2
CHHOCHOC22H44), 3.73 (dt, J = 9.6, 6.9 Hz, 2 H, 2 CHHOthp),
3.83–3.91 (m, 2 H, 2 CHHOCHOC22H44), 4.57 (dd, J = 4.5, 2.8
Hz, 2 H, 2
2
as white solid (11.55 g, 64%). H NMR (500
Hadfield, UK) and δ-octalactone (10a; Alfa Aesar, Ward Hill,
δ
1.22–1.63 (m, 48 H, CH2), 1.66–1.75 (m, 2
CHHCHO), 3.38 (dt, J =
USA), all chemicals were purchased from Sigma Aldrich Co.
(Steinheim, Germany) and were used without further purifica-
tion. All solvents were dried and distilled before use. The puri-
ty of all compounds was checked by thin-layer chromatog-
raphy (TLC) using silica gel 60 F254 plates (Macherey-Nagel
GmbH & Co KG, Düren, Germany). The chromatograms were
developed by means of bromothymol blue. Purification of all
×
×
×
×
×
×
×
-CHO-); ESI-MS m/z 533.42 (M + Na). Analytical
data are in accordance to data described previously.45
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ARTICLE
1,22-Dibromodocosane (3a).45 Triphenylphosphane (26.08 g,
99.4 mmol) was dissolved in dry dichloromethane (200 mL)
and bromine (15.89 g, 99.4 mmol), diluted in dichloromethane
(10 mL), was added dropwise into the solution whilst stirring at
acid (30%, 40 mL). The layers were separated and the aqueous
phase was extracted three times with diethyl ether (200 mL).
The combined ethereal phases were washed with brine (300
ml), dried over sodium sulphate and evaporated. The crude
product was purified by column chromatography with the use
of heptane/chloroform (2/8, v/v) as eluent yielding the diol as
a white solid.
(5RS)-Octane-1,5-diol (12a).61 Following the general proce-
dure, 10a (10.31 g, 72.5 mmol) and lithium aluminium hydride
(3.20 g, 84.2 mmol) gave 12a (7.64 g, 73%) as colourless oil. 1H
0 °C. Compound
2 (11.55 g, 22.6 mmol), dissolved in 100 mL
dichloromethane, was added and the mixture was stirred
overnight at room temperature. Afterwards, the organic layer
was washed with water (400 mL) and the crude dibromide 3a
was purified by column chromatography with heptane as elu-
ent yielding 3a as white crystals (9.0 g, 85%). 1H NMR (400
NMR (400 MHz, CDCl3, 27 °C)
1.66 (m, 10 H, CH2), 1.96 (s, 2 H, 2
CHOH, CH2OH); 13C NMR (100 MHz, CDCl3, 27 °C)
δ
0.85–0.98 (m, 3 H, CH3), 1.24–
OH), 3.55–3.67 (m, 3 H,
14.07 (CH3),
MHz, CDCl3, 27 °C)
(m, 4 H, 2 CH2CH2Br), 3.41 (t, J = 6.9 Hz, 4 H, 2
NMR (100 MHz, CDCl3, 27 °C) 28.17 (CH2(CH2)2Br), 28.75
δ
1.22–1.48 (m, 36 H, (CH2)18), 1.79–1.91
×
×
×
CH2Br); 13C
δ
δ
18.81 (CH3CH2), 21.76 (CH2(CH2)2OH), 32.51 (CH2CH2OH), 36.91
(CH2(CH2)3OH), 39.70 (CH3CH2CH2-), 62.57 (CH2OH), 71.47
(CHOH); ESI-MS m/z 169.55 (M + Na). Analytical data are in
accordance with data published previously.61
(5RS)-Tetradecane-1,5-diol (12b).62 Following the general
procedure, 10b (25.33 g, 111.9 mmol) and lithium aluminium
(CH2(CH2)3Br), 29.42, 29.52, 29.60, 29.63 and 29.67 (CH2),
32.84 (CH2CH2Br), 34.01 (CH2Br). Analytical data are in accord-
ance to data described previously.45
Synthesis of the secondary bromide 9 from the corresponding
lactone
hydride (4.68 g, 123.2 mmol) gave 12b (25.08 g, 97%) as a
1
white solid. H NMR (400 MHz, CDCl3, 27 °C)
The synthetic procedures and analytical data of compounds 5–
δ 0.88 (t, J = 6.9
8
can be found in the Electronic Supplementary Material
(ESI ).
2-{[(5RS)-5-Bromononyl]oxy}tetrahydro-2H-pyran (9). The
alcohol (0.84 g, 3.76 mmol) was dissolved in dry dichloro-
Hz, 3 H, CH3), 1.21–1.70 (m, 22 H, CH2), 3.57–3.63 (m, 1 H,
CHOH), 3.66 (t, J = 6.4 Hz, 2 H, CH2OH); 13C NMR (100 MHz,
†
CDCl3, 27 °C)
δ 14.07 (CH3), 21.81 (CH2(CH2)2OH), 22.65
8
(CH3CH2), 25.64 (CH3(CH2)6CH2), 29.29–29.67 (CH2), 31.87
(CH3CH2CH2), 32.62 and 32.63 (CH2CH2OH), 36.99 and 37.00
(alkylCHOHCH2), 37.54 (CH3(CH2)7CH2), 62.78 and 62.80
(CH2OH), 71.86 (CHOH); ESI-MS m/z 253.69 (M + Na), 269.65
(M + K). Analytical data are in accordance with data published
previously.62
methane (100 mL) at room temperature, 3,4-dihydro-2H-pyran
(0.62 g, 7.37 mmol) and pyridinium p-toluenesulfonate (0.95 g,
3.8 mmol) were added and the mixture was stirred for 20 h at
room temperature. Afterwards, the organic solution was
washed with water (150 mL), dried over sodium sulphate and
concentrated to dryness under reduced pressure. The crude oil
was purified by column chromatography using hep-
(12RS)-Octadecane-1,12-diol (12c).63 Following the general
procedure, 11 (13.07 g, 43.5 mmol) and lithium aluminium
tane/triethylamine/diethyl ether (98.5/0.5/1, v/v/v) yielding
9
hydride (1.98 g, 51.6 mmol) gave 12c (8.16 g, 66%) as a white
1
as colourless oil (1.09 g, 94%). C14H27BrO2 requires C, 54.72; H,
8.86; found: C, 54.14; H, 8.64; 1H NMR (400 MHz, CDCl3, 27 °C)
solid. M.p. 79–80 °C; H NMR (400 MHz, CDCl3, 27 °C)
δ
0.84–
-OH), 3.53–
3.61 (m, 1 H, CHOH), 3.64 (t, J = 6.7 Hz, 2 H, CH2OH); 13C NMR
(100 MHz, CDCl3, 27 °C) 14.06 (CH3), 22.60 (CH3CH2), 25.60
0.93 (m, 3 H, CH3), 1.25–1.65 (m, 32 H, CH2 and 2
×
δ
0.91 (t, J = 7.2 Hz, 3 H, CH3) 1.23–1.93 (m, 18 H, CH2) 3.34–
3.43 (m, 1 H, CHHOthp), 3.46–3.54 (m, 1 H, alkylOCHOCHH),
3.69–3.80 (m, H, CHHOthp), 3.82–3.92 (m, H, al-
kylOCHOCHH), 4.03 (quin, J = 6.4 Hz, 1 H, CHBr), 4.57 (t, J = 3.4
Hz, 1 H, OCHO); 13C NMR (100 MHz, CDCl3, 27 °C)
13.92 (CH3),
δ
1
1
and 25.62 (CH2CH2(CHOH)CH2CH2), 25.71 (CH2(CH2)2OH),
29.36– 29.67 (CH2), 31.83 (CH2CH2OH), 32.80 (CH3CH2CH2),
37.48 and 37.50 (CH2(CHOH)CH2), 63.08 (CH2OH), 72.01
(CHOH); ESI-MS m/z 309.93 (M + Na). Analytical data are in
accordance with data described previously.63
δ
19.64 and 19.66 (alkylOCHCH2CH2), 22.13 (CH3CH2), 24.34
(CH2(CH2)2Othp), 25.44 and 25.47 (alkylOCHOCH2CH2), 29.12
and 29.14 (alkylOCHCH2), 29.69 (CH3CH2CH2), 30.68 and 30.74
(CH2CH2Othp), 38.85 (CH3(CH2)2CH2), 38.93 (alkylCHBrCH2),
58.56 (CHBr), 62.32 and 62.35 (alkylOCHOCH2), 67.26 and
67.29 (CH2Othp), 98.84 and 98.90 (OCHO); ESI-MS m/z 329.56
(M + Na, 79Br isotope), 331.76 (M + Na, 81Br isotope).
General procedure for the synthesis of secondary tosylates 13
The diol 12 (1 equiv. 21.70 mmol) was dissolved in dry chloro-
form (150 mL). Afterwards, pyridine (2.4 equiv.) and trityl
chloride (1.2 equiv.) were added and the mixture was stirred
for 24 h at room temperature. After TLC shows the complete
conversion of the diol, tosyl chloride (1.2 equiv.) was added
and the mixture was stirred for further 72 h. For work-up,
water (100 mL) was added and the mixture was stirred for 1 h.
Afterwards, the mixture was extracted for several times with
chloroform. The combined organic phases were washed with
brine, dried over sodium sulphate and evaporated. The result-
ing crude product was diluted in cold heptane and the insolu-
ble trityl alcohol was separated by filtration. This procedure
has to be repeated until a clear solution of the crude product
General procedure for the synthesis of alkane-1,5-diols 12
Lithium aluminium hydride (1.1 equiv.) was suspended in dry
diethyl ether (200 mL) in a 1 L round-bottomed flask under
argon atmosphere. The suspension was cooled to −10 °C and
stirred for 1 h. The corresponding lactone 10 or hydroxy fatty
acid 11 (1 equiv.), dissolved in dry Et2O (50 mL), was added
very slowly. The ice bath was removed and the mixture was
stirred for 20 h at room temperature. Afterwards, the reaction
mixture was hydrolysed with ice and acidified using sulphuric
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ARTICLE
Organic and Biomolecular Chemistry
in heptane is obtained. Finally, the crude product was purified 1.18–1.67 (m, 20 H, CH2), 1.80–1.95 (m, 2 H, CH2CH2Br), 3.42
by MPLC using heptane and chloroform as eluents and the (t, J = 6.8 Hz, 2 H, CH2Br), 3.55–3.65 (m, 1 H, CH); 13C NMR (100
gradient technique.
MHz, CDCl3, 27 °C) δ 14.08 (CH3), 22.65 (CH2CH3), 24.31
1-[(4RS)-4-(Trityloxy)butyl]decyl 4-methylbenzenesulfonate (CH2(CH2)2Br), 25.61 (CH2(CH2)6CH3), 29.29–29.65 (CH2), 31.87
(13b). Following the general procedure, 12b (5.06 g, 22.0 (CH2CH2CH3), 32.77 (CH2CH2Br), 33.71 (CH2Br), 36.43 and 37.53
mmol), trityl chloride (7.28 g, 26.1 mmol), tosyl chloride (4.97 (CH2CHOHCH2), 71.69 (CH); APCI-MS m/z 275.1 (M – H2O + H,
g, 26.1 mmol), and pyridine (4.20 mL, 52.1 mmol) gave 13b 79Br isotope), 277.1 (M – H2O + H, 81Br isotope).
(10.49 g, 77%) as slight yellow oil. 1H NMR (400 MHz, CDCl3, 27
°C) 0.91 (t, J = 6.9 Hz, 3 H, CH2CH3), 1.13–1.69 (m, 22 H, CH2), procedure, 12c (8.16 g, 28.5 mmol), triphenylphosphane (7.81
2.40 (s, 3 H, CH3), 3.01 (t, J = 6.4 Hz, 2 H, CH2O), 4.56 (quin, J = g, 29.8 mmol) and tetrabromomethane (9.91 g, 29.9 mmol)
6.0 Hz, 1 H, CHO), 7.19–7.47 and 7.74–7.83 (m, 19 H, 3 C6H5, gave 14c (7.24 g, 73%) as white solid. M.p. 49–50 °C; C18H37BrO
C6H4); 13C NMR (100 MHz, CDCl3, 27 °C)
14.12 (CH2CH3), 21.57 requires C, 61.88; H, 10.67; found: C, 62.20; H, 10.75; H NMR
(CH3), 21.58 (CH2(CH2)2O), 22.68 (CH2CH3), 24.63 (400 MHz, CDCl3, 27 °C) 0.82–0.94 (m, 3 H, CH3), 1.24–1.50
(7RS)-18-Bromooctadecan-7-ol (14c). Following the general
δ
×
1
δ
δ
(CH3(CH2)6CH2), 29.28–29.71 (CH2), 31.88 (CH3CH2CH2), 33.99 (m, 28 H, CH2), 1.85 (m, 2 H, CH2CH2Br), 3.40 (t, J = 6.9 Hz, 2 H,
and 34.15 (CH2(CHO)CH2), 63.14 (CH2O), 84.35 (CHO), 86.34 CH2Br), 3.52–3.66 (m, 1 H, CH); 13C NMR (100 MHz, CDCl3, 27
(CH(C6H5)3), 126.85, 127.67, 127.70, 128.66, 129.60, 134.77, °C)
δ
14.06 (CH3), 22.60 (CH2CH3), 25.60 and 25.63
(CH2CH2CHOHCH2CH2), 28.16 (CH2(CH2)2Br), 28.74
4-methylbenzene- (CH2(CH2)3Br), 29.36–29.68 (CH2), 31.83 (CH2CH2Br), 32.82
and 144.38 (CH); ESI-MS m/z 649.73 (M + Na).
1-Hexyl-[(12RS)-12-(trityloxy)dodecyl]
sulfonate (13c). Following the general procedure, 12c (7.35 g, (CH2CH2CH3), 34.01 (CH2Br), 37.48 and 37.50 (CH2CHOHCH2),
25.7 mmol), trityl chloride (8.60 g, 30.9 mmol), tosyl chloride 72.00 (CH); APCI-MS m/z 331.3 (M – H2O + H, 79Br isotope),
(5.95 g, 31.2 mmol), and pyridine (4.97 mL, 61.6 mmol) gave 333.2 (M – H2O + H, 81Br isotope).
1
13c (2.96 g, 17%) as slight yellow oil. H NMR (400 MHz, CDCl3,
General procedure for the synthesis of thp-protected bromoalka-
27 °C)
2.43 (s, 3 H, CH3), 3.05 (t, J = 6.6 Hz, 2 H, CH2O), 4.54 (quin, J =
6.0 Hz, 1 H, CHO), 7.18–7.49 and 7.75–7.83 (m, 19 H, 3 C6H5, The bromoalkanol 14 (1 equiv.) was dissolved in dry dichloro-
C6H4); 13C NMR (100 MHz, CDCl3, 27 °C)
14.02 (CH2CH3), 21.57 methane (100 mL) at room temperature. 3,4-Dihydro-2H-
δ 0.85–0.89 (m, 3 H, CH2CH3), 1.11–1.72 (m, 30 H, CH2),
nols 15
×
δ
(CH3), 22.47 (CH2CH3), 24.63 and 24.69 (CH2CH2CHOCH2CH2), pyran (2.1 equiv.) and pyridinium p-toluenesulfonate (10
26.27 (CH2(CH2)2O), 28.93–30.06 (CH2), 31.57 (CH3CH2CH2), mol%) were added and the mixture was stirred for 20 h. Af-
34.12 and 34.14 (CH2CHOCH2), 63.70 (CH2O), 84.62 (CHO), terwards, the organic solution was washed with water (150
86.25 (CH(C6H5)3), 126.75, 127.64, 127.70, 128.68, 129.57, mL), dried over sodium sulphate and concentrated to dryness
134.85 and 144.53 (CH); ESI-MS m/z 705.43 (M + Na).
under reduced pressure. The crude oil was purified by column
chromatography using heptane/triethylamine/diethyl ether
(98.5/0.5/1, v/v/v) as eluent.
General procedure for the synthesis of bromoalkanols 14
The diol 12 (1 equiv. 5.0 g, 21.7 mmol), placed in a 250 mL
2-{[(1RS)-5-Bromo-1-propylpentyl]oxy}tetrahydro-2H-pyran
round-bottomed flask, was dissolved in dry CH2Cl2 (200 mL) (15a). Following the general procedure, 14a (6.09 g, 29.1
and cooled to 10 °C. Triphenylphosphane (1.1 equiv.) and mmol) and 3,4-dihydro-2H-pyran (4.57 g, 87.2 mmol) gave 15a
1
tetrabromomethane (1.1 equiv.) were added and the mixture (8.19 g, 96%) as colourless oil. H NMR (400 MHz, CDCl3, 27 °C)
was stirred for 20 h at 10 °C. Afterwards, the solvent was
evaporated. The crude product was purified by column chro- = 6.8, 3.6 Hz, 2 H, CH2Br), 3.44–3.52 (m, 1 H, OCHOCHH), 3.56–
matography using heptane/diethyl ether (8/2, v/v) as eluent. 3.68 (m, 1 H, CHOthp), 3.85–3.97 (m, 1 H, OCHOCHH), 4.59–
(4RS)-8-Bromooctan-4-ol (14a). Following the general pro- 4.67 (m, 1 H, OCHO); 13C NMR (100 MHz, CDCl3, 27 °C)
14.23
δ 0.86–0.96 (m, 3 H, CH3), 1.22–1.94 (m, 16 H, CH2), 3.41 (td, J
δ
cedure, 12a (7.54 g, 51.6 mmol), triphenylphosphane (14.96 g, and 14.31 (CH3), 18.35 and 18.88 (CH2CH3), 19.95 and 20.11
57.0 mmol) and tetrabromomethane (18.91 g, 57.0 mmol) (OCHO(CH2)2CH2), 23.61 and 24.21 (CH2(CH2)2Br), 25.51 and
gave 14a as colourless oil (6.21 g, 58%). 1H NMR (400 MHz, 25.52 (OCHOCH2CH2), 31.20–34.13 (CH2), 35.92 and 37.09
CDCl3, 27 °C)
δ 0.83–0.96 (m, 3 H, CH3), 1.23–1.64 (m, 8 H, (CH2CH2CH3), 62.73 and 62.95 (OCHOCH2), 76.15 and 76.36
CH2), 1.78–1.94 (m, 3 H, CH2CH2Br, CHOH), 3.38 (m, 2 H, (CHOthp), 97.56 and 97.91 (OCHO); ESI-MS m/z 315.30 (M +
CH2Br), 3.51–3.63 (m, 1 H, CH); 13C NMR (100 MHz, CDCl3, 27 Na, 79Br isotope), 317.22 (M + Na, 81Br isotope).
°C)
δ
(CH2CH2Br), 33.74 (CH2Br), 36.40 (CH2CHOH(CH2)2CH3), 39.63 (15b). Following the general procedure, 14b (3.85 g, 13.1
14.06 (CH3), 18.77 (CH2CH3), 24.29 (CH2(CH2)2Br), 32.76
2-{[(1RS)-1-(4-brombutyl)decyl]oxy}tetrahydro-2H-pyran
(CH2CH2CH3), 71.32 and 71.33 (CH); APCI-MS m/z 191.0 (M – mmol) and 3,4-dihydro-2H-pyran (2.37 g, 28.2 mmol) gave 15b
H2O + H, 79Br isotope), 193.0 (M – H2O + H, 81Br isotope).
1
(4.71 g, 95%) as colourless oil. H NMR (400 MHz, CDCl3, 27 °C)
(5RS)-1-Bromotetradecan-5-ol (14b). Following the general δ 0.79–0.90 (m, 3 H, CH3), 1.22–1.94 (m, 28 H, CH2), 3.35–3.42
procedure, 12b (5.0 g, 21.7 mmol), triphenylphosphane (5.87 (m, 2 H, CH2Br), 3.43–3.51 (m, 1 H, OCHOCHH), 3.59 (quin, J =
g, 22.3 mmol) and tetrabromomethane (7.58 g, 22.9 mmol) 4.6 Hz, 1H, CHOthp), 3.84–3.94 (m, 1 H, OCHOCHH), 4.58–4.66
gave 14b (4.40 g, 70%) as white solid. M.p. 31–32 °C; (m, 1 H, OCHO); 13C NMR (100 MHz, CDCl3, 27 °C)
δ 14.07
C14H29BrO requires C, 57.33; H, 9.97; found: C, 56.97; H, 9.87; (CH3), 19.91 and 20.08 (OCHO(CH2)2CH2), 22.65 (CH2CH3),
1H NMR (400 MHz, CDCl3, 27 °C)
δ
0.83–0.92 (m, 3 H, CH3), 23.60, 24.21, 25.03, 25.51, 25.53, 25.58 29.28, 29.30, 29.53,
10 | Org. Biomol. Chem., 2018, 00, 1-3
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ARTICLE
29.57, 29.59, 29.79, 29.84, 31.18, 31.22, 31.86, 31.87, 32.49, yielding the bis(tetrahydropyranyl)ethers 16 as waxy substanc-
32.95, 32.96, 33.55, 33.66, 33.76, 34.06, 34.96, 62.65 and es.
62.90 (OCHOCH2), 76.40 and 76.44 (CHOthp), 97.60 and 97.75
2,2’-[(4RS,35RS)-Octatriacontane-4,35-diylbis(oxy)]bis-
(OCHO); ESI-MS m/z 399.56 (M + Na, 79Br isotope), 401.52 (M + (tetrahydro-2H-pyran) (16a). Following the general procedure
Na, 81Br isotope).
variation A, 15a (7.85 g, 26.8 mmol), magnesium turnings (0.98
g, 40.3 mmol) and 3a (4.29 g, 9.2 mmol) gave 16a (2.95 g, 44%
2-{[(1RS)-12-Bromo-1-hexyldodecyl]oxy}tetrahydro-2H-
pyran (15c). Following the general procedure, 14c (7.20 g, 20.6 for the reaction at room temperature) as white solid. M.p. 51–
mmol) and 3,4-dihydro-2H-pyran (3.28 g, 39.0 mmol) gave 15c 52 °C; C48H94O4 requires C, 78.41; H, 12.89; found: C, 78.38; H,
(8.61 g, 97%) as colourless oil. H NMR (400 MHz, CDCl3, 27 °C) 12.77; 1H NMR (400 MHz, CDCl3, 27 °C)
1
δ
0.86–0.96 (m, 6 H, 2
×
δ
0.80–0.89 (m, 3 H, CH3), 1.22–1.73 (m, 34 H, CH2), 1.76–1.86 CH3), 1.22–1.87 (m, 80 H, CH2), 3.48 (dt, J = 10.7, 5.3 Hz, 2 H,
(m, 2 H, CH2CH2Br), 3.36 (t, J = 6.9 Hz, 2 H, CH2Br), 3.39–3.50 OCHOCHH), 3.60 (p, J = 5.4 Hz, 2 H, 2 CHOthp), 3.87–3.96
(m, 1 H, OCHOCHH), 3.56 (quin, J = 5.8 Hz, 1 H, CHOthp), 3.83– (m, 2 H, 2 OCHOCHH), 4.62–4.82 (m, 2 H, 2
OCHO); 13C NMR
3.93 (m, 1 H, OCHOCHH), 4.59–4.65 (m, 1 H, OCHO); 13C NMR (100 MHz, CDCl3, 27 °C)
14.27 and 14.35 (CH3), 18.32, 18.93,
2×
×
×
×
δ
(100 MHz, CDCl3, 27 °C) δ 14.04 and 14.06 (CH3), 19.91 and 19.97 and 19.99 (OCHO(CH2)2CH2, CH2CH3), 25.01, 25.57 and
19.94 (OCHO(CH2)2CH2), 22.59 and 22.63 (CH2CH3), 24.97 and 25.63 (OCHOCH2CH2, CH2CH2CHOthpCH2CH2), 29.60, 29.62,
24.98 (CH2CH2CHOthpCH2CH2), 25.75 (OCHOCH2CH2), 28.14 29.65, 29.67, 29.69, 29.84, 29.61, 31.21 and 31.24
and 28.15 (CH2CH2CH2Br), 28.73 (CH2(CH2)3Br), 29.39, 29.48, (OCHO(CH2)3CH2), 33.52, 35.09, 35.87 and 37.28
29.49, 29.51, 29.55, 29.79, 29.86, 31.20, 31.83, 32.81, 32.82, (CH2CHOthpCH2) 62.66 and 62.68 (OCHOCH2), 76.40 (CHOthp),
33.46, 33.49, 33.79, 35.00, 35.02, 62.53 and 62.73 (OCHOCH2), 97.39 and 97.57 (OCHO); ESI-MS m/z 742.11 (M + Li), 757.82
76.62 and 76.65 (CHOthp), 97.36 and 97.42 (OCHO); ESI-MS (M + Na).
m/z 455.90 (M + Na, 79Br isotope), 458.03 (M + Na, 81Br iso-
tope).
2,2’-[(7RS,38RS)-Tetratetracontane-7,38-diylbis(oxy)]bis-
(tetrahydro-2H-pyran (16b). Following the general procedure
variation B, 3b (1.95 g, 7.2 mmol), magnesium turnings (0.52 g,
21.6 mmol) and 15c (7.67 g, 17.7 mmol) gave 16b (3.41 g, 59%
for the reaction at room temperature) as white solid. M.p. 53–
General procedure for the synthesis of thp-protected 1,32-alkyl
branched 1,32-diols 16 via Grignard bis-coupling reaction
Variation A: Magnesium turnings (1.5 equiv.) were placed in a 54 °C; C54H106O4 requires C, 79.15; H, 13.04; found: C, 78.97; H,
100 mL round-bottomed flask under argon atmosphere. A 13.35; 1H NMR (400 MHz, CDCl3, 27 °C)
solution 15a or 15b (1 equiv.) in dry THF (20 mL) was added CH3), 1.23–1.90 (m, 92 H, CH2), 3.42–3.51 (m, 2 H, 2
dropwise. After the exothermic reaction had subsided, the OCHH), 3.59 (p, J = 5.8 Hz, 2 H, 2
mixture was stirred at 55 °C for 3 h. Afterwards, the excess OCHOCHH), 4.60–4.70 (m, 2 H, 2
magnesium was removed under argon atmosphere and the MHz, CDCl3, 27 °C) 14.06 and 14.08 (CH3), 19.95
δ
0.84–0.92 (m, 6 H, 2
×
×
OCH-
×
CHOthp), 3.86–3.98 (m, 2 H,
OCHO); 13C NMR (100
2×
×
δ
Grignard solution was cooled to 0 °C. A freshly prepared solu- (OCHO(CH2)2CH2), 22.65 and 22.67 (CH2CH3), 24.99, 25.01,
tion of dilithium tetrachlorocuprate(II) (0.1 M in THF, 0.1 25.58, 25.61 and 25.64 (OCHOCH2CH2, CH2CH2CHOthpCH2CH2),
equiv.) was added with stirring. After a solution of 2a (0.35– 29.51, 29.58, 29.61, 29.62, 29.65, 29.69, 29.84, 29.90, 31.22,
0.55 equiv.) in dry THF (70 mL) was added in one portion, stir- 31.86 (CH2CH2CH3), 33.49 (OCHO(CH2)3CH2), 35.05 and 35.41
ring was continued for 20 h at room temperature or at 0 °C.
Variation B: Magnesium turnings (3 equiv.) were placed in a (OCHO); ESI-MS m/z 826.18 (M + Li), 841.88 (M + Na).
100 mL round-bottomed flask under argon atmosphere. A 2,2’-[(10RS,41RS)-Pentacontane-10,41-diylbis(oxy)]bis-
(CH2CHOthpCH2) 62.62 (OCHOCH2), 76.71 (CHOthp), 97.43
solution 3b (1 equiv.) in dry THF (20 mL) was added dropwise. (tetrahydro-2H-pyran) (16c). Following the general procedure
After the exothermic reaction had subsided, the mixture was variation A, 15b (4.52 g, 12.0 mmol), magnesium turnings (0.44
stirred at 55 °C for 3 h. Afterwards, the excess magnesium was g, 18.1 mmol) and 3a (2.79 g, 6.0 mmol) gave 16c (0.70 g, 13%
removed under argon atmosphere and the Grignard solution for the reaction at 0 °C) as white solid. M.p. 56–58 °C;
was cooled to 0 °C. A freshly prepared solution of dilithium C60H118O4 requires C, 79.75; H, 13.16; found: C, 79.39; H, 13.38;
tetrachlorocuprate(II) (0.1 M in THF, 0.1 equiv.) was added 1H NMR (400 MHz, CDCl3, 27 °C)
with stirring. After a solution of 15c (2.5 equiv.) in dry THF (70 1.23–1.91 (m, 104 H, CH2), 3.44–3.52 (m, 2 H, 2
mL) was added in one portion, stirring was continued for 20 h 3.59 (p, J = 5.8 Hz, 2 H, 2 CHOthp), 3.86–3.98 (m, 2 H, 2
CHOCHH), 4.65 (t, J = 3.7 Hz, 2 H, 2
OCHO); 13C NMR (100
14.08 (CH3), 19.96 (OCHO(CH2)2CH2),
δ
0.84–0.92 (m, 6 H, 2
OCHOCHH),
O-
× CH3),
×
×
×
at room temperature or at 0 °C.
×
Both variations: For work-up, diethyl ether (150 mL) was MHz, CDCl3, 27 °C)
δ
added and the resulting mixture was poured into an ice-cold 22.67 (CH2CH3), 25.01, 25.58 and 25.64 (OCHOCH2CH,
saturated solution of ammonium chloride (150 mL). The organ- CH2CH2CHOthpCH2CH2), 29.31, 29.32, 29.60, 29.61, 29.62,
ic layer was separated and the aqueous phase was extracted 29.65, 29.69, 29.84, 29.90, 31.22, 31.89 (CH2CH2CH3), 33.48
several times with diethyl ether. The combined ethereal phas- (OCHO(CH2)3CH2), 35.03 (CH2CHOthpCH2) 62.62 (OCHOCH2),
es were washed with brine, dried over sodium sulphate and 76.69 (CHOthp), 97.42 (OCHO); ESI-MS m/z 926.65 (M + Na).
concentrated to dryness under reduced pressure. The crude
General procedure for the synthesis of 1,32-alkyl branched 1,32-
product was purified by column chromatography using hep-
diols 17
tane/triethylamine/diethyl ether (98/0.5/1.5, v/v/v) as eluent
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Organic and Biomolecular Chemistry
The bis(tetrahydropyranyl)ether 16 and catalytic amounts of fied by MPLC using chloroform/methanol/water and the gradi-
pyridinium p-toluenesulfonate were suspended in dry metha- ent technique.
nol (50 mL) and the mixture was heated under reflux for at
least 3 h until a white precipitate appeared and no educt is methylammonio)ethyl phosphate] (PC-C32(1,32C3)-PC). Fol-
detectable via TLC. The hot suspension was filtered off. lowing the general procedure, 17a (1.00 g, 1.76 mmol) gave
(4RS,35RS)-Octatriacontane-4,35-diol (17a). Following the PC-C32(1,32C3)-PC (0.17 g, 11%). 1H NMR (400 MHz,
general procedure, 16a (2.95 g, 4.0 mmol) gave 17a (2.01 g, CDCl3/CD3OD, 27 °C) 0.80–0.91 (m, 6 H, 2 x CH3), 1.16–1.57
89%) as white solid. M.p. 109–111 °C; 1H NMR (400 MHz, (m, 68 H, CH2), 3.13–3.18 (m, 18 H, 2
N(CH3)3), 3.46–3.56 (m,
CDCl3, 27 °C) 0.92–0.99 (m, 6 H, 2 CH3), 1.26–1.59 (m, 68 H, 4 H, 2 OCH2CH2N), 4.07–4.21 (m, 6 H, 2 OCH2CH2N, 2 CHO);
CH2), 3.58–3.64 (m, 2 H, 2 13.90 and 13.93
CHOH); 13C NMR (125 MHz, CDCl3, 13C-NMR (100 MHz, CDCl3/CD3OD, 27 °C):
50 °C) 14.00 (CH3), 18.75 (CH2CH3), 25.59 (CH3), 18.10 (CH2CH3), 24.97 (CH2CH2CHO(CH2)2CH3), 29.57,
(1RS,32RS)-1,32-Dipropyldotriacontane-1,32-diylbis[2-tri-
δ
×
×
δ
×
×
×
×
×
δ
δ
(CH2CH2CHOH(CH2)2CH3), 29.55, 29.56, 29.59, 29.60, 29.62, 29.60 and 29.79 (CH2), 35.12 and 35.16 (CH2CHO(CH2)2CH3),
37.54 (CH2CHOH(CH2)2CH3), 39.71 (CH2CH2CH3), 71.71 (CHOH); 37.16 and 37.20 (CH2CH2CH3), 54.09 (N(CH3)3), 58.58 and 58.63
APCI-MS m/z 531.6 (M – 2
×
H2O + H).
(OCH2CH2N), 66.54 (OCH2CH2N), 76.76 and 76.83 (CHO); ESI-
(7RS,38RS)-Tetratetracontane-7,38-diol (17b). Following the MS m/z 897.66 (M + H), 919.84 (M + Na); HRMS m/z calcd. for
general procedure, 16b (3.41 g, 4.2 mmol) gave 17b (2.38 g, C48H104N2O8P2 (M + 2H) 449.3629; found: 499.3648; calcd. for
88%) as white solid. M.p. 111–113 °C; 1H NMR (500 MHz, C48H103N2O8P2 (M + H) 897.7184; found: 897.7223; HPLC tR
=
CDCl3, 50 °C)
80 H, CH2), 3.55–3.70 (m, 2 H, 2
CDCl3, 50 °C) 13.92 (CH3), 22.53 (CH2CH3), 25.55 and 25.60 methylammonio)ethyl phosphate] (PC-C32(1,32C6)-PC). Fol-
δ
0.90 (t, J = 6.5 Hz, 6 H, 2
×
CH3), 1.22–1.54 (m, 7.3 min, purity: 98.3 %.
×
CHOH); 13C NMR (125 MHz,
(1RS,32RS)-1,32-Dihexyldotriacontane-1,32-diylbis[2-tri-
δ
(CH2CH2CHOHCH2CH2), 29.31, 29.55, 29.56, 29.59, 29.61, lowing the general procedure, 17b (0.20 g, 0.31 mmol) gave
29.62, 29.67, 31.79 (CH2CH2CH3), 37.52 (CH2CHOHCH2), 71.99 PC-C32(1,32C6)-PC (0.10 g, 33%). 1H NMR (500 MHz,
(CHOH); APCI-MS m/z 615.7 (M – 2
(10RS,41RS)-Pentacontane-10,41-diol (17c). Following the (m, 80 H, CH2), 3.18 (s, 18 H, 2
general procedure, 16c (0.70 g, 0.78 mmol) gave 17c (0.50 g, OCH2CH2N), 4.12–4.23 (m, 6 H, 2
88%) as white solid. M.p. 115–116 °C; 1H NMR (500 MHz, NMR (125 MHz, CDCl3/CD3OD, 27 °C):
CDCl3, 50 °C) 0.90 (t, J = 6.5 Hz, 6 H, 2 CH3), 1.05–1.56 (m, 22.52 (CH2CH3), 24.86 and 24.92 (CH2CH2CHOCH2CH2), 29.38,
92 H, CH2), 3.55–3.65 (m, 2 H, 2
CHOH); 13C NMR (125 MHz, 29.54, 29.60 and 29.76 (CH2), 31.76 (CH2CH2CH3), 34.96, 34.99
CDCl3, 50 °C) 13.94 (CH3), 22.58 (CH2CH3), 25.60 and 35.01 (CH2CHOCH2), 54.12 (N(CH3)3), 58.75 and 58.78
×
H2O + H).
CDCl3/CD3OD, 27 °C)
δ
0.82–0.89 (m, 6 H, 2
N(CH3)3), 3.53–3.59 (m, 4 H, 2
OCH2CH2N, 2
CHO); 13C
13.83 and 13.87 (CH3),
× CH3), 1.20–1.62
×
×
×
×
δ
δ
×
×
δ
(CH2CH2CHOHCH2CH2), 29.24, 29.50, 29.56, 29.62, 29.67, 31.83 (OCH2CH2N), 66.50 and 66.51 (OCH2CH2N), 76.96, 77.21 and
(CH2CH2CH3), 37.51 (CH2CHOHCH2), 71.98 (CHOH); APCI-MS 77.47 (CHO); ESI-MS m/z 981.81 (M + H), 1003.78 (M + Na);
m/z 699.7 (M – 2
×
H2O + H).
HRMS m/z calcd for C54H115N2O8P2 (M + H) 981.8123; found:
981.8111; HPLC tR = 4.9 min, purity: 95.8 %.
General procedure for the synthesis of bis(phosphocholines)
(1RS,32RS)-1,32-Dinonyldotriacontane-1,32-diylbis[2-tri-
2-Bromoethylphosphoric acid dichloride (8 equiv.) was poured methylammonio)ethyl phosphate] (PC-C32(1,32C9)-PC). Fol-
into dry chloroform (15 mL) whilst cooling with ice/water. A lowing the general procedure, 17c (0.50 g, 0.68 mmol) gave
mixture of dry triethylamine (14 equiv.) in dry chloroform (15 PC-C32(1,32C9)-PC (0.15 g, 21%). 1H NMR (500 MHz,
mL) was added slowly with stirring, which was continued for CDCl3/CD3OD, 27 °C)
further 30 min at 0 °C. Diol 17 (1 equiv.) was poured in one 1.37 (m, 84 H, CH2), 1.48–1.58 (m, 8 H, 2
portion into the mixture. To dissolve the solid diol, the reac- (s, 18 H, 2 N(CH3)3), 3.55–3.61 (m, 4 H, 2
tion mixture was slowly heated to 50 °C until a clear solution 4.21 (m, 6 H, 2 OCH2CH2N, 2
appeared and was stirred for 24 h at 50 °C. Afterwards, the CDCl3/CD3OD, 27 °C): 13.90 (CH3), 22.56 (CH2CH3), 24.94
δ 0.84 (t, J = 6.9 Hz, 6 H, 2× CH3), 1.18–
×
CH2CHOCH2), 3.19
×
× OCH2CH2N), 4.11–
×
×
CHO); 13C NMR (125 MHz,
δ
mixture cooled to room temperature and stirred for further 72 (CH2CH2CHOCH2CH2), 29.24, 29.55, 29.60, 29.62, 29.78 and
h. After the complete conversion of the diol, crushed ice was 29.79 (CH2), 31.81 (CH2CH2CH3), 34.98 and 35.01 (CH2CHOCH2),
added and the mixture was stirred vigorously for 2 h. The 54.13 (N(CH3)3), 58.76 and 58.80 (OCH2CH2N), 66.49
organic layer was separated and the aqueous phase was ex- (OCH2CH2N), 77.12 and 77.17 (CHO); ESI-MS m/z 1065.98 (M +
tracted several times with chloroform (3 x 50 mL). The com- H), 1087.96 (M + Na); HRMS m/z calcd for C60H128N2O8P2 (M +
bined organic phases were concentrated under reduced pres- 2H) 533.4568; found: 533.4564; calcd for C60H127N2O8P2 (M +
sure, and the oily brownish residue was dissolved in THF/water H) 1065.9062; found: 1065.9051; HPLC tR = 3.4 min, purity:
(9/1, v/v, 20 mL). After 1 h of stirring, the solvent was evapo- 96.4 %.
rated and the crude bromo esters were transferred into a
Physicochemical characterisation
mixture of dry chloroform (30 mL), dry acetonitrile (30 mL). A
ethanolic solution of trimethylamine (20 equiv.) was added Sample preparation. The bolalipid was suspended in H2O (Mil-
slowly, and the mixture was kept in a closed tube while warm- li-Q Millipore water). Homogeneous suspensions were ob-
ing up to 50 °C for 48 h. The solution was allowed to stand for tained by heating and vortexing.
7 days at room temperature. Afterwards, the reaction mixture
was evaporated to dryness and the crude bolalipid was puri- performed using a MicroCal VP-DSC differential scanning calo-
DSC. As described previously,64 DSC measurements were
12 | Org. Biomol. Chem., 2018, 00, 1-3
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Organic & Biomolecular Chemistry
ARTICLE
rimeter (MicroCal Inc. Northampton, MA, USA). Before the
measurements, the sample suspension (c = 1 mg mL−1) and the
water reference were degassed under vacuum while stirring. A
heating rate of 60 K h−1 was used, and the measurements were
performed in the temperature interval from 5 °C to 90 °C. To
check the reproducibility, three consecutive scans were rec-
orded for each sample. The water/water baseline was sub-
tracted from the thermogram of the sample, and the DSC
scans were evaluated using MicroCal Origin 8.0 software.
Turbidity measurements. Transmission measurements were
performed using a Litesizer 500 (Anton Paar GmbH, Graz, Aus-
tria). The sample suspension (c = 1 mg mL−1 in water) was set
in a low volume quartz cuvette (Hellma Analytics, Müllheim,
Germany) and the measurement was performed in the tem-
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TOC-graphic
The syntheses of three single-chain, alkyl-branched bolalipids—using a Grignard bis-coupling
reaction as key step—and first investigations of the lyotropic behaviour of these lipids are
reported.