Synthesis of enantiomerically pure α-hydroxylated nervonic acid - A chiral pool approach to α-hydroxylated unsaturated fatty acids

Article abstract of DOI:10.1055/s-0033-1341276

A scalable and robust synthesis to α-hydroxylated nervonic acid is presented. The chiral-pool approach starts with malic acid and employs only highly reliable reactions such as Wittig reactions, protecting group conversions, and reductions. Depending on the configuration of malic acid being used as starting material both enantiomers can be obtained. Georg Thieme Verlag Stuttgart. New York.

Full text of DOI:10.1055/s-0033-1341276

LETTER
▌1435
lSetyternthesis of Enantiomerically Pure α-Hydroxylated Nervonic Acid – A Chiral
Pool Approach to α-Hydroxylated Unsaturated Fatty Acids
Synthesis of Enantiomerically Pure α-Hydroxylated Nervonic Acid
Martin Pawliczek, Jan Wallbaum, Daniel B. Werz*
Technische Universität Braunschweig, Institut für Organische Chemie, Hagenring 30, 38106 Braunschweig, Germany
Fax +49(531)3915272; E-mail: d.werz@tu-braunschweig.de
Received: 04.03.2014; Accepted after revision: 31.03.2014
fatty acid 5 (Scheme 1, b).10–12 Such a highly electron-rich
Abstract: A scalable and robust synthesis to α-hydroxylated ner-
species is easily attacked by molecular oxygen which is
vonic acid is presented. The chiral-pool approach starts with malic
bubbled through the solution. The drawback of this meth-
od is that a racemic mixture of 6 is obtained. Therefore,
acid and employs only highly reliable reactions such as Wittig reac-
tions, protecting group conversions, and reductions. Depending on
the configuration of malic acid being used as starting material both we became interested to design a simple and scalable
enantiomers can be obtained.
route to access enantiomerically pure α-hydroxylated cis-
unsaturated fatty acids starting from a compound of the
chiral pool.13 Our idea was to use malic acid, an α-hydrox-
ylated diacid, which is commercially available in both en-
antiomeric forms.
Key words: lipids, fatty acids, malic acid, nervonic acid
Biological membranes act as boundaries between the inte-
rior and the exterior of living cells. In most eukaryotes
they consist of a lipid bilayer. Although the bilayer pos-
sesses a fluid character the constituents commonly do not
mix uniformly. Inner and outer leaflet of the lipid bilayer
differ strongly, and in addition different types of lipids
segregate in the plane of the membrane driven by lipid–
lipid interactions.1 As a result domains (lipid rafts) are
formed. These rafts are postulated to be of crucial impor-
tance in a plethora of cellular functions such as signaling,
endocytic traffic, and adhesion of viruses, bacteria, and
other cells.2–4 Depending on the type and the percentage
of different lipids (sphingomyelin, cholesterol, glyco-
sphingolipids) in the mixture the fate and the dynamics of
the rafts heavily change. In order to investigate how rafts
are formed and evolve on a molecular level prerequisite is
access to pure compounds. It is well-known that hydrox-
ylated fatty acids in glycosphingolipids change their func-
tion significantly.5–7 One idea is that a hydroxyl group
next to the carbonyl group enhances exposure of the car-
bohydrate part on the cell surface.8 Due to the enzymatic
machinery which is responsible for hydroxylations only
one of two possible stereoisomers is formed during this
process.
a) Approach to α-hydroxylated saturated fatty acids
(in enantiomerically pure form)
BnO
Li
R
+
O
2
1
O
[Red.]
[Ox.]
BnO
HO
R
OH
R
OH
3
4
b) Approach to α-hydroxylated unsaturated fatty acids
(as racemic mixture)
1) n-BuLi
(2 equiv)
O
O
HO
R
HO
R
n
n
2) O2
OH
rac-6
5
Scheme 1 Approaches to α-hydroxylated fatty acids
The synthesis of α-hydroxylated nervonic acid started
with the transformation of D-malic acid to the correspond-
ing aldehyde 8 by a literature-known sequence consisting
of protection, reduction, and oxidation (Scheme 2).14 Af-
terwards, the alkyl chain was elongated via Wittig reac-
tion using phosphonium salt 9.
In order to investigate biophysical questions in detail one
relies on pure compounds being not isolated, but synthe-
sized by chemical means. In the case of hydroxylated sat-
urated fatty acids of type 4 one usually takes advantage of
a ring opening of an enantiomerically pure epoxide 1 by a
lithiated acetylide 2 (Scheme 1, a).9 Later, the triple bond
of 3 gets reduced to an ethano unit. However, this method
is not suitable for unsaturated hydroxylated fatty acids
since reduction would also eliminate the double bond. An-
other approach to reach α-hydroxylation is based on oxi-
dation of the carboxylate enolate of the corresponding
This phosphonium salt containing the required eleven car-
bons as well as a terminal hydroxyl group for further
transformations was prepared starting from 11-bromoun-
decanol (7) by benzyl protection followed by subsequent
formation of the Wittig salt using triphenylphosphine as
described in the literature.15
With both starting materials in hand, the first olefination
was performed using NaHMDS in THF to obtain the
desired product 10 in 62% yield. Hydrogenation of the
double bond by Pd(OH)2 on charcoal under hydrogen at-
mosphere removes the benzyl protecting group in the
same step and furnishes the corresponding primary alco-
SYNLETT 2014, 25, 1435–1437
Advanced online publication: 13.05.2014
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0033-1341276; Art ID: st-2014-b0192-l
© Georg Thieme Verlag Stuttgart · New York

1436
M. Pawliczek et al.
LETTER
hol, which was readily oxidized with pyridinium chloro- problems utilizing a chiral stationary phase. Therefore,
chromate (PCC) to afford aldehyde 11 within 30 minutes. the enantiomeric purity of α-hydroxylated nervonic acid
The latter was used without further purification in the fi- (14) was determined by a coupling experiment using the
nal Z-selective Wittig reaction to accomplish the fatty acid enantiomerically pure chiral amine 15 (Scheme 3).
chain of α-hydroxy nervonic acid. The coupling between HATU-mediated coupling in the presence of Hünig base16
phosphonium salt 12 with NaHMDS as base (leading to produced only one observable diastereomer of 16 as prov-
an unstabilized phosphorus ylide) and aldehyde 11 yield- en by 13C NMR spectroscopy of the crude reaction mix-
ed the protected Z-configured fatty acid 13 in 23% overall ture, wherefore an enantiomeric ratio of >95:5 was
yield from 10, whereas only traces of the E-configured obtained. The use of the crude mixture is necessary to en-
product could be observed which were easily removed by sure that a potentially occurring diastereomer is not re-
silica gel column chromatography. Final deprotection of moved by column chromatography.
13 was achieved using a mixture of 1 M aq HCl in THF to
obtain the free α-hydroxy nervonic acid 14 in 96% yield.
O
Scheme 2 depicts the synthetic route to the R-configured
+
HO
NH2
7
11
congener. In addition we prepared also the S-configured
enantiomer starting from L-malic acid in a completely
analogous way with similar yields.
OH
15
14
HATU, i-Pr2NEt
In order to prove that our transformations did not lead to
(partial) racemization of the stereocenter we performed an
HPLC experiment. However, we came across separation
THF, DMF, 0 °C, 2 h
O
N
11
7
O
H
OH
OH
OH
10
HO
Br
16
7
OH
D-malic acid
3 steps
O
Scheme 3 HATU-mediated formation of 16 to determine the enan-
tiomeric purity of α-hydroxylated nervonic acid (14)
2 steps
ref. 15
In summary, we developed a fast and efficient synthetic
method to prepare α-hydroxylated nervonic acid (14) in
five steps from readily available aldehyde 8. Prerequisite
was a chiral-pool approach using malic acid as starting
material combined with the choice of suitable protecting
groups. Depending on the configuration of malic acid
both enantiomers of α-hydroxylated nervonic acid can be
obtained. The presented approach should be applicable to
access a variety of enantiomerically pure unsaturated fatty
acids with an α-hydroxyl group, even such with more than
one unsaturation. Studies incorporating nervonic acid into
complex glycolipids to study their phase behavior in
membranes are under way and will be reported in due
course.
ref. 14
O
H
OBn
10
O
Br Ph3P
O
O
9
8
a
BnO
O
O
b, c
10
O
11
O
O
O
O
10
11
d
(R,Z)-5-[13-(Benzyloxy)tridec-2-en-1-yl]-2,2-dimethyl-1,3-diox-
olan-4-one (10)
O
O
e
To a suspension of phosphonium bromide 9 (724 mg, 1.20 mmol,
1.00 equiv) in THF (4 mL) was added NaHMDS (1 M in THF, 1.20
mL, 1.20 mmol, 1.00 equiv) at 0 °C and further stirred for 1 h at r.t.
Afterwards, the reaction mixture was cooled to –78 °C, and alde-
hyde 8 (190 mg, 1.20 mmol, 1.20 equiv) dissolved in THF (2 mL)
was added dropwise. The reaction mixture was allowed to warm to
r.t. overnight. Then H2O was added, the phases were separated, and
the aqueous layer was extracted with Et2O. The combined organic
layers were washed with brine, dried over Na2SO4, and the solvent
was removed in vacuo. The residue was purified by column chro-
matography on silica gel (pentane–EtOAc = 40:1 to 20:1) to obtain
the title compound (301 mg, 749 μmol, 62%) as a colorless oil.
11
HO
11
7
7
O
O
OH
14
13
Br Ph3P
12
7
Scheme 2 Synthesis of α-hydroxylated nervonic acid from malic
acid. Reagents and conditions: a) 9, NaHMDS, THF, 0 °C to r.t., 1 h;
then 9, –78 °C to r.t., 16 h, 62%; b) Pd(OH)2/C, H2, MeOH, r.t., 16 h;
c) PCC, CH2Cl2, 0 °C to r.t., 1 h; d) 12, NaHMDS, THF, 0 °C to r.t.,
1 h; then 11, –78 °C to r.t., 16 h, 23% (over 3 steps); e) THF–HCl, 60 °C,
96%; THF = tetrahydrofuran, NaHMDS = sodium bis(trimethylsi-
lyl)amide.
1H NMR (200 MHz, CDCl3): δ = 1.25–1.40 (m, 14 H), 1.54 (d,
J = 0.6 Hz, 3 H), 1.57–1.66 (m, 5 H), 2.05 (q, J = 7.0 Hz, 2 H),
2.42–2.72 (m, 2 H), 3.46 (t, J = 6.7 Hz, 2 H), 4.44 (dd, J = 6.6, 4.6
Hz, 1 H), 4.50 (s, 2 H), 5.32–5.47 (m, 1 H), 5.52–5.68 (m, 1 H),
Synlett 2014, 25, 1435–1437
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Enantiomerically Pure α-Hydroxylated Nervonic Acid
1437
7.27–7.36 (m, 5 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 26.0,
26.2, 27.1, 27.4, 29.3, 29.4, 29.4, 29.5, 29.5, 29.5, 29.6, 29.8, 70.5,
72.8, 74.1, 110.6, 122.1, 127.4, 127.6, 128.3, 134.5, 138.7, 172.8
ppm. IR (ATR): ν = 2993, 2925, 2853, 1794, 1455, 1271, 1113 cm–1.
ESI-HRMS: m/z calcd for C25H38O4 [M + Na]+: 425.26623; found:
425.26662.
78.0 μmol, 1.20 equiv) in THF (0.5 mL) was added HATU (32.0
mg, 85 μmol, 1.30 equiv) dissolved in DMF (0.5 mL) at 0 °C, and
the mixture was stirred at this temperature for 2 h. Then brine and
aq HCl (4:1) were added, and the aqueous phase was extracted three
times with EtOAc. The combined organic phases were dried over
Na2SO4, and the solvent was removed in vacuo to obtain the crude
coupling product 16. To prove the enantiomeric purity, 1H and 13
C
(R,Z)-5-(Docos-13-en-1-yl)-2,2-dimethyl-1,3-dioxolan-4-one
(13)
NMR spectra were recorded using the crude product mixture.
1H NMR (300 MHz, CDCl3): δ = 0.88 (t, J = 6.3 Hz, 3 H), 1.21–
1.40 (m, 32 H), 1.50 (d, J = 1.5 Hz, 3 H), 1.53–1.67 (m, 1 H), 1.74–
1.88 (m, 1 H), 2.06 (q, J = 5.8 Hz, 4 H), 4.11 (dd, J = 7.8, 3.7 Hz, 1
H), 5.12 (dd, J = 7.8, 7.0 Hz, 1 H), 5.35 (t, J = 5.1 Hz, 2 H), 6.80 (d,
J = 6.8 Hz, 1 H), 7.27–7.36 (m, 5 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 14.1, 21.9, 24.9, 27.2, 29.3, 29.3, 29.3, 29.3, 29.4, 29.4,
29.5, 29.5, 29.5, 29.6, 29,6, 29.6, 29.7, 29.8, 29.8, 31.9, 34.9, 39.5,
48.4, 72.1, 126.0, 127.3, 128.6, 129.9, 143.0, 172.9 ppm. ESI-
HRMS: m/z calcd for C34H55O2N [M + Na]+: 508.41250; found:
508.41238.
To a solution of 10 (427 mg, 1.06 mmol, 1.00 equiv) in MeOH (10
mL) was added Pd(OH)2/C (20 wt%, 100 mg, 100 μmol, 10 mol%),
and the atmosphere was changed to H2 and stirred for 16 h at r.t. The
suspension was filtered through a short pad of Celite and eluted with
additional MeOH. The solvents were removed in vacuo. The resi-
due was dissolved in anhydrous CH2Cl2 (10 mL), cooled to 0 °C and
PCC (434 mg, 2.02 mmol, 1.90 equiv) was added. After 10 min at
0 °C the reaction was stirred for additional 20 min at r.t., filtered
through a short pad of Celite and eluted with additional CH2Cl2. Af-
terwards, activated carbon was added to the filtrate and stirred for
further 30 min before the mixture was filtered through a short pad
of silica to obtain the corresponding crude aldehyde 11 (172 mg) af-
ter concentration, which was used without further purification.
Acknowledgment
Financial support by the German Research Foundation (SFB 803
‘Functionality controlled by organization in and between mem-
branes’) is greatly acknowledged. M.P. is grateful to the Fonds der
Chemischen Industrie for his Doktorandenstipendium. D.B.W.
thanks the German Research Foundation (Emmy Noether and
Heisenberg Fellowships) and the Fonds der Chemischen Industrie
(Dozentenstipendium).
To a suspension of phosphonium bromide 12 (285 mg, 600 μmol,
0.57 equiv) in THF (6 mL) was added NaHMDS (1 M in THF, 0.60
mL, 600 μmol, 0.57 equiv) at 0 °C, and the resulting mixture was
stirred for an additional hour at r.t. Then, the solution was cooled to
–78 °C, and the crude aldehyde 11 dissolved in THF (3 mL) was
added dropwise. The resulting mixture was stirred for 10 min at
–78 °C and then 30 min at r.t. Afterwards, silica (300 mg) was add-
ed, the solvent was removed in vacuo, and the residue was purified
by column chromatography on silica (pentane–EtOAc = 150:1) to
obtain the title compound (105 mg, 23% over three steps) as a col-
orless oil.
Supporting Information for this article is available online
at
http://www.thieme-connect.com/products/ejournals/journal/
10.1055/s-00000083.SunogIpimrfiantoSuIpg
n
fonirtat
ori
1H NMR (600 MHz, CDCl3): δ = 0.88 (t, J = 7.0 Hz, 3 H), 1.24–1.36
(m, 30 H), 1.38–1.58 (m, 2 H), 1.54 (d, J = 0.5 Hz, 3 H), 1.60 (d,
J = 0.5 Hz, 3 H), 1.68–1.75 (m, 1 H), 1.84–1.90 (m, 1 H), 2.01 (q,
J = 6.7 Hz, 4 H), 4.39 (dd, J = 7.2, 4.3 Hz, 1 H), 5.35 (t, J = 5.1 Hz,
2 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 14.1, 22.7, 24.9, 25.8,
27.2, 27.2, 27.2, 29.2, 29.3, 29.3, 29.3, 29.4, 29.5, 29.5, 29.6, 29.6,
29.6, 29.6, 29.8, 29.8, 31.6, 31.9, 74.1, 110.4, 129.9, 129.9, 173.4
ppm. IR (ATR): ν = 2998, 2922, 2853, 1797, 1462, 1383, 1265,
1125 cm–1. ESI-HRMS: m/z calcd for C27H50O3 [M + Na]+:
445.36522; found: 445.36553.
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(–)-(R)-α-Hydroxynervonic Acid (14)
Protected acid 13 was dissolved in a mixture of THF (4 mL) and
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NaCl was added until saturation of the aqueous phase was observed.
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over Na2SO4. After removal of the solvent in vacuo the title com-
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1H NMR (600 MHz, CDCl3): δ = 0.88 (t, J = 7.2 Hz, 3 H), 1.22–
1.36 (m, 30 H), 1.39–1.52 (m, 2 H), 1.67–1.74 (m, 1 H), 1.83–1.90
(m, 1 H), 2.01 (q, J = 5.7 Hz, 4 H), 4.78 (q, J = 3.3 Hz, 1 H), 5.34–
5.36 (m, 2 H) ppm. We could neither detect the proton of the car-
boxylic acid nor the one of the hydroxyl group. 13C NMR (125
MHz, CDCl3): δ = 14.1, 22.7, 24.8, 27.2, 27.2, 29.3, 29.3, 29.3,
29.3, 29.3, 29.4, 29.5, 29.6, 29.6, 29.7, 29.7, 29.8, 29.8, 31.9, 34.2,
70.2, 129.9, 129.9, 178.9 ppm. IR (ATR): ν = 3543, 3163, 2917,
2848, 1709, 1664, 1464, 1207, 1092 cm–1. ESI-HRMS: m/z calcd
for C24H46O3 [M + Na]+: 405.33392; found: 405.33405. [α]D21.5 –2.5
(c 1.76, CHCl3).
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(16)
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amine 15 (8.40 μL, 65.0 μmol, 1.00 equiv), and i-Pr2NEt (13.6 μL,
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© Georg Thieme Verlag Stuttgart · New York
Synlett 2014, 25, 1435–1437