Efficient synthesis of the very-long-chain n-3 fatty acids, tetracosahexaenoic acid (C24:6n-3) and tricosahexaenoic acid (C 23:6n-3)

Article abstract of DOI:10.1007/s11745-011-3541-5

Tetracosahexaenoic acid (C₂₄:6n-3, THA, 3) is an essential biosynthetic precursor in mammals of docosahexaenoic acid (C₂₂:6n-3, DHA, 1), the end-product of the metabolism of n-3 fatty acids. THA 3 is present in commercially valuable fishes, such as flathead flounder. Tricosahexaenoic acid (C₂₃:6n-3, TrHA, 2), an odd-numbered-chain fatty acid, has been identified from marine organisms such as the dinoflagellate, Amphidinium carterae. To date, few studies have examined THA 3 and TrHA 2 due to difficulties in detecting and identifying these compounds, so their chemical and biological properties remain poorly characterized. Only one methodology for the chemical synthesis of THA 3 has been presented, and no method for the synthesis of TrHA 2 has been reported. We report here the efficient synthesis of THA 3 in four steps in 56% overall yield, and the synthesis of TrHA 2 in six steps in 48% overall yield. We also present the synthesis of Δ²-THA 4, an intermediate of β-oxidation of THA 3 to DHA 1, in three steps in 73% overall yield.

Full text of DOI:10.1007/s11745-011-3541-5

Lipids (2011) 46:455–461
DOI 10.1007/s11745-011-3541-5
METHODS
Efficient Synthesis of the Very-Long-Chain n-3 Fatty Acids,
Tetracosahexaenoic Acid (C24:6n-3) and Tricosahexaenoic
Acid (C23:6n-3)
•
Toshimasa Itoh Ayako Tomiyasu
•
Keiko Yamamoto
Received: 8 January 2011 / Accepted: 4 February 2011 / Published online: 24 February 2011
Ó AOCS 2011
Abstract Tetracosahexaenoic acid (C24:6n-3, THA, 3) is
an essential biosynthetic precursor in mammals of doco-
sahexaenoic acid (C22:6n-3, DHA, 1), the end-product of
the metabolism of n-3 fatty acids. THA 3 is present in
commercially valuable fishes, such as flathead flounder.
Tricosahexaenoic acid (C23:6n-3, TrHA, 2), an odd-
numbered-chain fatty acid, has been identified from marine
organisms such as the dinoflagellate, Amphidinium carte-
rae. To date, few studies have examined THA 3 and TrHA
2 due to difficulties in detecting and identifying these
compounds, so their chemical and biological properties
remain poorly characterized. Only one methodology for the
chemical synthesis of THA 3 has been presented, and no
method for the synthesis of TrHA 2 has been reported. We
report here the efficient synthesis of THA 3 in four steps in
56% overall yield, and the synthesis of TrHA 2 in six steps
in 48% overall yield. We also present the synthesis of
D2-THA 4, an intermediate of b-oxidation of THA 3 to
DHA 1, in three steps in 73% overall yield.
Abbreviations
ALA a-Linolenic acid
DHA Docosahexaenoic acid
EPA
Eicosapentaenoic acid
THA Tetracosahexaenoic acid
TrHA Tricosahexaenoic acid
TAG Triacyl-sn-glycerol
Introduction
The polyunsaturated fatty acid, docosahexaenoic acid
(C22:6n-3, DHA, 1), is largely found in neuronal tissues
such as brain and retina [1]. DHA 1 is an essential com-
ponent of neuronal membranes, is a precursor of potent
neuroprotective mediators, and is important in helping to
prevent both cardiovascular disease and metabolic syn-
drome [2, 3]. Mammals obtain DHA 1 directly from dietary
sources, especially fish, but can also generate DHA 1 in the
liver from n-3 fatty acid precursors obtained from eating
plants [4–6]. The capacity of the human liver to produce
DHA 1 may become critical for maintaining normal levels
of DHA 1 in the brain and retina [4, 6–8] if insufficient
DHA 1 is available from the diet, as is often the case in
modern societies [9].
Keywords Tetracosahexaenoic acid Á Tricosahexaenoic
acid Á Docosahexaenoic acid Á Chemical synthesis Á
Alzheimer’s disease Á b-oxidation Á Biosynthetic precursor Á
Odd-numbered fatty acids Á Fish oil Á DHA metabolism
T. Itoh Á A. Tomiyasu Á K. Yamamoto (&)
Laboratory of Drug Design and Medicinal Chemistry, Showa
Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen,
Machida, Tokyo 194-8543, Japan
The pathway for DHA biosynthesis in the liver is shown
in Fig. 1. a-Linolenic acid (18:3n-3, ALA) is converted to
eicosapentaenoic acid (20:5n-3, EPA), which is converted
into docosapentaenoic acid (22:5n-3, DPA) and then into
tetracosahexaenoic acid (24:6n-3, THA, 3). Desaturase and
elongase enzymes are localized in the endoplasmic retic-
ulum of the hepatocyte and act on n-3 fatty acids. Desat-
urase progressively introduces double bonds, and elongase
e-mail: yamamoto@ac.shoyaku.ac.jp
A. Tomiyasu
Institute of Biomaterials and Bioengineering, Tokyo Medical
and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku,
Tokyo 101-0062, Japan
123

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Lipids (2011) 46:455–461
Fig. 2 Chemical structures of the fatty acids discussed in this paper.
Docosahexaenoic acid (1), tricosahexaenoic acid (2), tetracosahexa-
enoic acid (3) and D2-tetracosahexaenoic acid (4)
polyunsaturated fatty acids have also been identified from
marine organisms [21]. Odd-chain fatty acids can be used
in lipid metabolism [22]. The C23 fatty acid, tricosahexa-
enoic acid (C23:6n-3, TrHA 2), is found in the dinoflagel-
late, Amphidinium carterae [23]. To date, few studies have
examined THA 3 and TrHA 2 due to difficulties in
detecting and identifying these compounds, so their
chemical and biological properties remain poorly charac-
terized. To our knowledge, only one chemical synthesis
method for THA 3 has been reported [24, 25], and none for
TrHA 2. In the course of our synthetic and structural bio-
logical studies of polyunsaturated fatty acids [26–30], we
attempted to derive very-long-chain n-3 fatty acids from
DHA ester. In this paper, we report the efficient chemical
synthesis of THA 3, TrHA 2, and an intermediate of
b-oxidation of THA 3 to DHA 1, D2-THA 4 (Fig. 2).
Fig. 1 Metabolic pathway of bioconversion of n-3 polyunsaturated
fatty acids
extends shorter-chain n-3 fatty acids by two-carbon units,
thereby generating very-long-chain THA 3 [10]. THA 3 is
then transported into the peroxisomes and converted to
DHA 1 by b-oxidation reactions that involve several spe-
cific enzymes: acyl-coenzyme A oxidase, D-bifunctional
protein, and peroxisomal thiolases [11–14]. DHA 1, the
end-product of the metabolism of n-3 fatty acids, is
delivered to the brain by the circulatory system and accu-
mulates in the brain membranes. In contrast, THA 3 is not
incorporated into the brain membranes but is an essential
precursor of DHA 1. Recently, Piomelli’s group reported
that levels of DHA precursors, particularly ALA and THA
3, are elevated in the livers of patients with Alzheimer’s
disease, whereas expression of peroxisomal D-bifunctional
protein, which catalyzes the conversion of THA 3 to DHA
1, is reduced [15]. Piomelli et al. concluded that deficient
liver biosynthesis of DHA 1 correlates with cognitive
impairment in Alzheimer’s patients. Thus, the critical
importance of THA 3 in mammals is becoming clearer.
THA 3 is found in various marine organisms [16–18].
Nichols et al. [19] reported that THA 3 constitutes 9.3% of
the total fatty acids in the jellyfish Aurelia sp. Recently,
Tomita and Ando reported that THA 3 is found preferen-
tially at the sn-2 position (1.6–23.3 mol%) of triacyl-sn-
glycerols (TAG) from flathead flounder flesh [20]. Flathead
flounder is extensively consumed by humans; therefore,
we intake THA 3 in the form of TAG concentrating THA 3
at the sn-2 position. Odd-numbered very-long-chain
Materials and Methods
General Experimental Procedures
DHA ethyl ester 5 was a kind gift from the Maruha Nichiro
holdings (Tsukuba, Japan). All the other reagents were
purchased from commercial sources and used without fur-
ther purification. Organic solvents used were dried by
standard methods. All reactions were performed under a
nitrogen atmosphere. Silica gel (wako gel C200) was used
for column chromatography, and pre-coated silica gel
1
60F254 plates (0.25 mm, Merck) were used for TLC. H
NMR (300 MHz) and 13C NMR (75 MHz) were recorded
using a Bruker AV300 instrument in CDCl3 solution with
TMS as an internal standard and the chemical shifts are
given in d values. Splitting patterns are indicated as follows:
s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m,
multiplet. Mass spectra were recorded on a JEOL MS700
spectrometer using NBA as positive-ion FAB matrix.
(4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10,13,16,19-
Hexaenal (6)
To a stirred solution of DHA ethyl ester 5 (157 mg,
0.440 mmol) in CH2Cl2 (1 mL) was added 1.0 M
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Lipids (2011) 46:455–461
457
diisobutylaluminium hydride (DIBAL-H) in toluene
(523 lL, 0.523 mmol) at -78 °C and the mixture was
stirred for 1 h. The reaction was quenched with 1 N HCl
aq. and the mixture was extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4 and
evaporated. The residue was chromatographed on silica gel
(9 g, 5% EtOAc–hexane) to give aldehyde 6 (117 mg,
85%). A large scale preparation of 6 was performed using
10.1 g of DHA ester 5 under the similar conditions and
7.45 g of pure compound 6 was obtained (84% yield).
1H NMR (CDCl3) d: 9.78 (1 H, m, H-1), 5.33–5.44 (12
H, m, H-4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20), 2.78–2.92
(10 H, m, H-6, 9, 12, 15, 18), 2.51 (2 H, m, H-2), 2.40 (2 H,
m, H-3), 2.08 (2 H, quint, J = 7.5 Hz, H-21), 0.97 (3 H, t,
J = 7.5 Hz, H-22); 13C NMR (CDCl3) d: 201.9, 132.0,
129.4, 128.6, 128.4, 128.29, 128.27, 128.1 (2 carbons),
127.90, 127.86, 127.7, 127.0, 43.7, 25.64 (3 carbons),
25.59, 25.5, 20.6, 20.1, 14.3; HRMS (FAB): Calcd for
C22H33O [M ? H]?: 313.2531; found: 313.2537.
128.23, 128.18, 128.16, 128.09 (2 carbons), 127.9, 127.0,
119.7, 51.0, 28.8, 26.6, 25.6 (4 carbons), 25.5, 20.6, 14.3;
HRMS (FAB): Calcd for C25H37O2 [M ? H]?: 369.2794;
found: 369.2792.
Methyl (6Z,9Z,12Z,15Z,18Z,21Z)-Tetracosa-
6,9,12,15,18,21-Hexaenoate (8)
To a solution of 2E/2Z mixture 7 (32 mg, 87 lmol) in
MeOH (0.5 mL) was added Mg turnings (20 mg,
0.84 mmol) at 0 °C and the mixture was stirred for 3.5 h at
room temperature. After addition of H2O, the mixture was
extracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4 and evaporated. The residue was
chromatographed on silica gel (10 g, 1% EtOAc–hexane)
to give 8 (26 mg, 81%). A large scale preparation of 8 was
performed using 1.07 g of 2E/2Z mixture 7 under the
similar conditions and 820 mg of pure compound 8 was
1
obtained (76% yield). H NMR (CDCl3) d: 5.28–5.56 (12
H, m, H-6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22), 3.67 (3
H, s, H-CO2Me), 2.73–2.92 (10 H, m, H-8, 11, 14, 17, 20),
2.32 (2 H, m, H-2), 2.08 (4 H, m, H-5, 23), 1.65 (2 H, quint,
J = 7.7 Hz, H-3), 1.40 (2 H, quint, J = 7.7 Hz H-4), 0.98
(3 H, t, J = 7.5 Hz, H-24); 13C NMR (CDCl3) d: 174.1,
132.0, 129.7, 128.6, 128.4, 128.3, 128.19, 128.16, 128.12,
128.10, 128.0, 127.9, 127.0, 51.5, 34.0, 29.0, 26.9, 25.6 (4
carbons), 25.5, 24.6, 20.6, 14.3; HRMS (FAB): Calcd for
C25H39O2 [M ? H]?: 371.2950; found: 371.2954.
Methyl (2E,6Z,9Z,12Z,15Z,18Z,21Z)-Tetracosa-
2,6,9,12,15,18,21-Heptaenoate (7)
To a stirred suspension of sodium hydride (0.71 g,
29.6 mmol) in THF (6 mL) was added trimethyl phos-
phonoacetate (4.25 mL, 29.6 mmol) at 0 °C and the mix-
ture was stirred for 10 min. A solution of aldehyde 6
(4.63 g, 14.8 mmol) in THF (45 mL) was added to the
mixture. The reaction mixture was stirred at 0 °C for 1 h,
then quenched with water at 0 °C and extracted with
EtOAc. The organic layer was washed with brine, dried
over MgSO4 and evaporated. The residue was chromato-
graphed on silica gel (60 g, 0.5% EtOAc–hexane) to give
2Z-7 (0.39 g, 7%) and 2E-7 (4.71 g, 87%) in this order.
2E-7 Rf = 0.51 (10% EtOAc-hexane). 1H NMR
(CDCl3) d: 6.96 (1 H, dt, J = 15.6, 6.6 Hz, H-3), 5.84 (1
H, d, J = 15.6 Hz, H-2), 5.28–5.46 (12 H, m, H-6, 7, 9, 10,
12, 13, 15, 16, 18, 19, 21, 22), 3.73 (3 H, s, H-CO2Me),
2.78–2.91 (10 H, m, H-8, 11, 14, 17, 20), 2.25 (4 H, m,
H-4, 5), 2.08 (2 H, quint, J = 7.5 Hz, H-23), 0.97 (3 H, t,
J = 7.5 Hz, H-24); 13C NMR (CDCl3) d: 167.0, 148.6,
132.0, 129.1, 128.6, 128.29, 128.26, 128.22 (2 carbons),
128.08, 128.06, 128.02, 127.9, 127.0, 121.3, 51.4, 32.1,
25.7, 25.6 (3 carbons), 25.5, 20.6, 14.3; HRMS (FAB):
Calcd for C25H37O2 [M ? H]?: 369.2794; found:
(6Z,9Z,12Z,15Z,18Z,21Z)-Tetracosa-6,9,12,15,18,21-
Hexaenoic Acid (THA 3)
A solution of 8 (1.19 g, 3.23 mmol) in 5% KOH/MeOH–
H2O (19:1, 16 mL) was stirred at 50 °C for 1 h. The reaction
mixture was neutralized with 10% HCl aq. and extracted
with EtOAc. The organic layer was washed with water, dried
over MgSO4, and evaporated. The residue was chromato-
graphed on silica gel (110 g, 50% EtOAc–hexane) to give
THA 3 (1.01 g, 87 %). 1H NMR (CDCl3) d: 5.27–5.55 (12 H,
m, H-6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22), 2.74–2.91 (10
H, m, H-8, 11, 14, 17, 20), 2.36 (2 H, t, J = 7.4 Hz, H-2),
2.08 (4 H, m, H-5, 23), 1.66 (2 H, m, H-3), 1.42 (2 H, m, H-4),
0.98 (3 H, t, J = 7.5 Hz, H-24); 13C NMR (CDCl3) d: 179.9,
132.0, 129.6, 128.6, 128.4, 128.3, 128.22, 128.18, 128.1,
128.03, 127.96, 128.9, 127.0, 33.9, 29.0, 26.8, 25.6 (4 car-
bons), 25.5, 24.3, 20.6, 14.3; HRMS (FAB): Calcd for
C24H37O2 [M ? H]?: 357.2794; found: 357.2790.
1
369.2780. 2Z-7 Rf = 0.65 (10% EtOAc-hexane). H NMR
(CDCl3) d: 6.22 (1 H, dt, J = 11.5, 7.5 Hz, H-3), 5.79 (1
H, dt, J = 11.5, 1.7 Hz, H-2), 5.26–5.45 (12 H, m, H-6, 7,
9, 10, 12, 13, 15, 16, 18, 19, 21, 22), 3.70 (3 H, s,
H-CO2Me), 2.68–2.91 (12 H, m, H-4, 8, 11, 14, 17, 20),
2.22 (2 H, m, H-5), 2.07 (2 H, quint, J = 7.5 Hz, H-23),
0.97 (3 H, t, J = 7.5 Hz, H-24); 13C NMR (CDCl3) d:
166.7, 149.8, 132.0, 128.8 (2 carbons), 128.6, 128.25,
(2E,6Z,9Z,12Z,15Z,18Z,21Z)-Tetracosa-
2,6,9,12,15,18,21-Heptaenoic Acid (D2-THA 4)
A solution of 2E-7 (100 mg, 272 lmol) in 5% KOH/
iPrOH–H2O (19:1, 10 mL) was stirred at 50 °C for 3 h.
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Lipids (2011) 46:455–461
The reaction mixture was neutralized with 10% HCl aq.
and extracted with EtOAc. The organic layer was washed
with water, dried over MgSO4, and evaporated. The residue
was passed through a short silica gel column (1 g, 70%
EtOAc–hexane) to give D2-THA 4 (95 mg, 99 %). 1H
NMR (CDCl3) d: 7.07 (1 H, dt, J = 15.6, 6.6 Hz, H-3),
5.84 (1 H, d, J = 15.6 Hz, H-2), 5.28–5.46 (12 H, m, H-6,
7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22), 2.77–2.91 (10 H, m,
H-8, 11, 14, 17, 20), 2.20–2.36 (4 H, m, H-4, 5), 2.08 (2 H,
quint, J = 7.5 Hz, H-23), 0.97 (3 H, t, J = 7.5 Hz, H-24);
13C NMR (CDCl3) d: 171.9, 151.3, 132.0, 129.2, 128.6,
128.3 (2 carbons), 128.2, 128.13, 128.08 (2 carbons),
128.0, 127.9, 127.0, 121.1, 32.2, 25.6 (4 carbons), 25.5,
20.5, 14.3; HRMS (FAB): Calcd for C24H35O2 [M ? H]?:
355.2637; found: 355.2624.
2.75–2.87 (10 H, m, H-6, 9, 12, 15, 18), 2.44 (3 H, s, H-Ph-
Me), 2.04–2.12 (4 H, m, H-3, 21), 1.71 (2 H, quint,
J = 6.5 Hz, H-2), 0.97 (3 H, t, J = 7.5 Hz, H-22); 13C
NMR (CDCl3) d: 144.7, 133.2, 132.0, 129.8 (2 carbons),
129.4, 128.6, 128.29, 128.26 (2 carbons), 128.1 (2 car-
bons), 128.0, 127.9 (4 carbons), 127.0, 69.9, 28.8, 25.6 (3
carbons), 25.5 (2 carbons), 23.0, 21.6, 20.6, 14.3; HRMS
(FAB): Calcd for C29H41O3S [M ? H]?: 469.2776; found:
469.2777.
(5Z,8Z,11Z,14Z,17Z,20Z)-Tricosa-5,8,11,14,17,20-
Hexaenenitrile (11)
A mixture of tosylate 10 (6.71 g, 14.3 mmol) and KCN
(1.31 g, 20.1 mmol) in DMSO (40 mL) was stirred at
70 °C for 2.5 h. After addition of H2O at 0 °C, the mixture
was extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The residue
was chromatographed on silica gel (100 g, 5% EtOAc–
hexane) to give cyanide 11 (4.51 g, 97%). 1H NMR
(CDCl3) d: 5.32–5.40 (12 H, m, H-5, 6, 8, 9, 11, 12, 14, 15,
17, 18, 20, 21), 2.78–2.87 (10 H, m, H-7, 10, 13, 16, 19),
2.34 (2 H, t, J = 7.2 Hz, H-2), 2.01–2.14 (4 H, m, H-4,
22), 1.66 (2 H, m, H-3), 1.42 (2 H, m, H-3), 0.98 (3 H, t,
J = 7.5 Hz, H-23); 13C NMR (CDCl3) d: 132.0, 130.3,
128.6, 128.4, 128.3 (2 carbons), 128.1, 128.0, 127.9, 127.8,
127.3, 127.0, 119.6, 26.0, 25.7 (4 carbons), 25.5, 25.3,
20.6, 16.5, 14.3; HRMS (FAB): Calcd for C23H34N
[M ? H]?: 324.2691; found: 324.2696.
(4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10,13,16,19-
Hexaen-1-ol (9)
To a stirred solution of DHA ethyl ester 5 (6.17 g,
17.3 mmol) in CH2Cl2 (40 mL) was added 1.0 M DIBAL-H
in toluene (51.5 mL, 51.5 mmol) at 0 °C and the mixture
was stirred for 1.5 h. The mixture was quenched with 1 N
HCl aq. at 0 °C and extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4 and
evaporated. The residue was chromatographed on silica gel
(100 g, 10% EtOAc–hexane) to give alcohol 9 (5.06 g,
1
93%). H NMR (CDCl3) d: 5.28–5.47 (12 H, m, H-4, 5, 7,
8, 10, 11, 13, 14, 16, 17, 19, 20), 3.66 (2 H, t, J = 6.4 Hz,
H-1), 2.85 (10 H, m, H-6, 9, 12, 15, 18), 2.16 (2 H, quint,
J = 6.4 Hz, H-3), 2.08 (2 H, quint, J = 7.5 Hz, H-21),
1.65 (2 H, quint, J = 6.4 Hz, H-2), 0.98 (3 H, t,
J = 7.5 Hz, H-22); 13C NMR (CDCl3) d: 132.1, 129.4,
128.6, 128.5, 128.30, 128.27, 128.20, 128.16, 128.1 (2
carbons), 127.9, 127.0, 62.5, 32.5, 25.65 (2 carbons), 25.63,
25.60, 25.5, 23.6, 20.6, 14.3; HRMS (FAB): Calcd for
C22H35O [M ? H]?: 315.2688; found: 315.2686.
(5Z,8Z,11Z,14Z,17Z,20Z)-Tricosa-5,8,11,14,17,20-
Hexaenal (12)
To a stirred solution of cyanide 11 (2.53 g, 7.86 mmol) in
CH2Cl2 (1 mL) was added 1.0 M DIBAL-H in toluene
(10.1 mL, 10.1 mmol) at -20 °C and the mixture was
stirred for 1 h. The mixture was quenched with 10%
aqueous potassium sodium tartrate at 0 °C and the mixture
was extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4 and evaporated. The residue
was chromatographed on silica gel (55 g, 3% EtOAc–
hexane) to give aldehyde 12 (1.93 g, 75%). 1H NMR
(CDCl3) d: 9.87 (1 H, t, J = 1.6 Hz, H-1), 5.23–5.48 (12
H, m, H-5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21), 2.78–2.91
(10 H, m, H-7, 10, 13, 16, 19), 2.45 (2 H, td, J = 7.3 Hz,
1.6 Hz, H-2), 2.02–2.18 (4 H, m, H-4, 22), 1.71 (2 H, quint,
J = 7.3 Hz, H-3), 0.97 (3 H, t, J = 7.5 Hz, H-23); 13C
NMR (CDCl3) d: 202.4, 132.0, 129.1, 128.8, 128.6, 128.3,
128.23, 128.19, 128.12 (2 carbons), 128.08, 127.9, 127.0,
43.8, 26.9, 25.7 (2 carbons), 25.6 (2 carbons), 25.5, 21.9,
20.6, 14.3; HRMS (FAB): Calcd for C23H35O [M ? H]?:
327.2688; found: 327.2696.
(4Z,7Z,10Z,13Z,16Z,19Z)-Docosa-4,7,10,13,16,19-
Hexaen-1-yl 4-Methylbenzenesulfonate (10)
A mixture of alcohol 9 (5.06 g, 16.1 mmol) and p-toluene-
sulfonyl chloride (4.61 g, 24.2 mmol) in pyridine
(25 mL) was stirred at 0 °C for 13 h. After addition of
water, the mixture was extracted with EtOAc. The organic
layer was washed with 1 N HCl aq., saturated NaHCO3 aq.
and brine, dried over MgSO4 and evaporated. The residue
was chromatographed on silica gel (110 g, 5% EtOAc–
hexane) to give tosylate 10 (6.71 g, 89%). 1H NMR
(CDCl3) d: 7.79 (2 H, d, J = 8.3 Hz, H-Ar), 7.34 (2 H, d,
J = 8.3 Hz, H-Ar), 5.29–5.42 (12 H, m, H-4, 5, 7, 8, 10,
11, 13, 14, 16, 17, 19, 20), 4.03 (2 H, t, J = 6.5 Hz, H-1),
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Lipids (2011) 46:455–461
459
Methyl (5Z,8Z,11Z,14Z,17Z,20Z)-Tricosa-
5,8,11,14,17,20-Hexaenoate (13)
Results and Discussion
Two fatty acids, THA 3 and D2-THA 4, were synthesized
using DHA ethyl ester 5 as the starting material as shown in
Scheme 1. Ester 5 was reduced with 1.2 equivalents of
diisobutylaluminium hydride (DIBAL-H) at -78 °C to
afford aldehyde 6 in 85% yield. Aldehyde 6 was subjected
to the Horner–Wadsworth–Emmons reaction using
trimethyl phosphonoacetate [31] to give two-carbon elon-
gated a,b-unsaturated ester 7 in 94% yield as a mixture of
2E-isomer (87%) and 2Z-isomer (7%). These two isomers
were separated easily by silica gel column chromatography.
Regioselective hydrogenation of a,b-unsaturated ester 7
was achieved by reduction using Mg turnings in MeOH to
provide 8 in good yield (81%) [31–33]. Ester 8 was treated
with 5% KOH/MeOH–H2O (19:1) to provide desired
compound THA 3 in 87% yield. On the other hand, D2-THA
4 was obtained in 99% yield upon treatment of 2E-7 with
5% KOH/iPrOH–H2O (19:1). Thus, THA 3 was synthesized
in four steps in 56% overall yield and D2-THA 4 was syn-
thesized in three steps in 73% overall yield. Kuklev et al.
[24] reported the synthesis of THA 3 from DHA methyl
ester, via a malonic ester derivative as a precursor of two-
carbon-elongated fatty acid; he reported a 21% overall yield
achieved in five steps. Thus, our synthetic route is more
facile and efficient than the previously reported route.
TrHA 2 was synthesized from DHA ethyl ester 5 as
shown in Scheme 2. Ester 5 was reduced with DIBAL-H at
0 °C to afford alcohol 9 in 93% yield. Alcohol 9 was then
treated with TsCl to give tosylate 10 in 89% yield. Tosylate
10 was treated with KCN at 70 °C to afford one-carbon-
elongated cyanide 11 in 97% yield. Cyanide 11 was
reduced with DIBAL-H to afford aldehyde 12 in 75%
yield. Aldehyde 12 was converted to ester 13 in 91% yield
by alkaline-iodine oxidation, a reaction previously devel-
oped by our group [34]. Ester 13 was treated with
5% KOH/MeOH–H2O (19:1) to provide desired compound
TrHA 2 in 88% yield. Thus, TrHA 2 was synthesized in six
steps in 48% overall yield. To our knowledge, this is the
first report of the chemical synthesis of TrHA 2.
A mixture of aldehyde 12 (1.93 g, 5.92 mmol), KOH
(864 mg, 15.4 mmol) and I2 (1.95 g, 7.70 mmol) in MeOH
(75 mL) was stirred at 0 °C for 1 h. After addition of 10%
solution of aqueous Na2S2O3, the mixture was extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4 and evaporated. The residue was chro-
matographed on silica gel (40 g, 2% EtOAc–hexane) to
1
give methyl ester 13 (1.91 g, 91%). H NMR (CDCl3) d:
5.28–5.44 (12 H, m, H-5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20,
21), 3.67 (3 H, s, H-CO2Me), 2.76–2.90 (10 H, m, H-7, 10,
13, 16, 19), 2.32 (2 H, t, J = 7.4 Hz, H-2), 2.04–2.12 (4 H,
m, H-4, 22), 1.70 (2 H, quint, J = 7.4 Hz, H-3), 0.98 (3 H,
t, J = 7.5 Hz, H-23); 13C NMR (CDCl3) d: 174.0, 132.0,
128.9, 128.8, 128.6, 128.25, 128.24, 128.19, 128.15,
128.09 (2 carbons), 127.9, 127.0, 51.5, 33.4, 26.5, 26.63 (4
carbons), 25.5, 24.8, 20.6, 14.3; HRMS (FAB): Calcd for
C24H37O2 [M ? H]?: 357.2794; found: 357.2790.
(5Z,8Z,11Z,14Z,17Z,20Z)-Tricosa-5,8,11,14,17,20-
Hexaenoic Acid (TrHA 2)
A solution of ester 13 (1.19 g, 3.34 mmol) in 5% KOH/
MeOH–H2O (19:1, 16 mL) was stirred at 50 °C for 1 h.
The reaction mixture was neutralized with 10% HCl aq.
and extracted with EtOAc. The organic layer was washed
with water, dried over MgSO4, and evaporated. The residue
was chromatographed on silica gel (110 g, 50% EtOAc–
hexane) to give TrHA 2 (1.01 g, 88 %). 1H NMR (CDCl3)
d: 5.27–5.44 (12 H, m, H-5, 6, 8, 9, 11, 12, 14, 15, 17, 18,
20, 21), 2.76–2.91 (10 H, m, H-7, 10, 13, 16, 19), 2.37 (2
H, t, J = 7.4 Hz, H-2), 2.02–2.17 (4 H, m, H-4, 22), 1.70
(2 H, quint, J = 7.4 Hz, H-3), 0.97 (3 H, t, J = 7.5 Hz,
H-23); 13C NMR (CDCl3) d: 179.9, 132.0, 129.0, 128.8,
128.6, 128.27, 128.20, 128.19, 128.15 (2 carbons), 128.09,
127.9, 127.0, 33.4, 26.5, 25.6 (4 carbons), 25.5, 24.5, 20.6,
14.3; HRMS (FAB): Calcd for C23H35O2 [M ? H]?:
343.2637; found: 343.2643.
Scheme 1 Synthetic route from
DHA ester to
tetracosahexaenoic acid (3) and
D2-tetracosahexaenoic acid (4)
123

460
Lipids (2011) 46:455–461
Scheme 2 Synthetic route from
DHA ester to tricosahexaenoic
acid (2)
4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent
of a 4-desaturase. J Biol Chem 266:19995–20000
In conclusion, we have developed efficient chemical
methods for the synthesis of the very-long-chain n-3
polyunsaturated fatty acids, THA 3, D2-THA 4 and TrHA
2. Using these synthetic compounds, we are now investi-
gating their biological activities and metabolism.
12. Sprecher H, Luthria DL, Mohammed BS, Baykousheva SP
(1995) Reevaluation of the pathways for the biosynthesis of
polyunsaturated fatty acids. J Lipid Res 36:2471–2477
13. Moore SA, Hurt E, Yoder E, Sprecher H, Spector AA (1995)
Docosahexaenoic acid synthesis in human skin fibroblasts
involves peroxisomal retroconversion of tetracosahexaenoic acid.
J Lipid Res 36:2433–2443
14. Su HM, Moser AB, Moser HW, Watkins PA (2001) Peroxisomal
straight-chain Acyl-CoA oxidase and D-bifunctional protein are
essential for the retroconversion step in docosahexaenoic acid
synthesis. J Biol Chem 276:38115–38120
Acknowledgments We are grateful to Prof. Hirokazu Tamamura of
Tokyo Medical and Dental University for support of this project. Part
of this work was supported by a Grant-in-Aid for Young Scientist (no.
80536110) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.
15. Astarita G, Jung KM, Berchtold NC, Nguyen VQ, Gillen DL,
Head E, Cotman CW, Piomelli D (2010) Deficient liver biosyn-
thesis of docosahexaenoic acid correlates with cognitive impair-
ment in Alzheimer’s disease. PLoS ONE 5:e12538
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