A Facile and Efficient Method for the Synthesis of Labeled and Unlabeled Very Long Chain Polyunsaturated Fatty Acids

Article abstract of DOI:10.1002/aocs.12484

Several methods are available for elongation of fatty acid acyl chains. The present paper describes adaptation to the fatty acid field of a previously published protocol for manganese-based Wurtz type coupling of alkyl bromides. 22-Bromo-3(Z),6(Z),9(Z),12(Z),15(Z),18(Z)-docosahexaene, easily prepared from 4(Z),7(Z),10(Z),13(Z),16(Z),19(Z)-docosahexaenoic acid, was coupled to homologous ω-bromoesters by stirring for 4 hours at 40°C in the presence of manganese powder, a nickel catalyst and terpyridine. This afforded in yields of 70–75% a series of ω3-hexaenoates of chain lengths of 32–40 carbons. The corresponding fatty acids of >98% purity were obtained following saponification and final purification. By using methyl [2,2,3,3,4,4-²H₆]10-bromodecanoate as coupling partner it was possible to prepare a very long chain fatty acid in isotopically labeled form, i.e., [2,2,3,3,4,4-²H₆]14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-dotriacontahexaenoic acid. Also prepared were the monounsaturated long chain fatty acids 15(Z)-octadecenoic acid and 15(Z)-tetracosenoic acid. Very long chain fatty acids have been isolated from retina and other tissues and are of biological relevance. The methodology described will assist in further analytical and biological studies in this field.

Full text of DOI:10.1002/aocs.12484

J Am Oil Chem Soc (2021)
DOI 10.1002/aocs.12484
ORIGINAL ARTICLE
A Facile and Efficient Method for the Synthesis of Labeled and
Unlabeled Very Long Chain Polyunsaturated Fatty Acids
Mats Hamberg1
Received: 18 June 2020 / Revised: 7 February 2021 / Accepted: 15 March 2021
© 2021 The Author. Journal of the American Oil Chemists' Society published by Wiley Periodicals LLC on behalf of American Oil Chemists'
Society.
Abstract Several methods are available for elongation of
fatty acid acyl chains. The present paper describes adaptation
to the fatty acid field of a previously published protocol for
manganese-based Wurtz type coupling of alkyl bromides.
22-Bromo-3(Z),6(Z),9(Z),12(Z),15(Z),18(Z)-docosahexaene,
easily prepared from 4(Z),7(Z),10(Z),13(Z),16(Z),19(Z)-
docosahexaenoic acid, was coupled to homologous
ω-bromoesters by stirring for 4 hours at 40ꢀC in the presence
of manganese powder, a nickel catalyst and terpyridine. This
afforded in yields of 70–75% a series of ω3-hexaenoates of
chain lengths of 32–40 carbons. The corresponding fatty acids
of >98% purity were obtained following saponification and
final purification. By using methyl [2,2,3,3,4,4-2H6]
10-bromodecanoate as coupling partner it was possible to pre-
pare a very long chain fatty acid in isotopically labeled form,
Keywords Fatty acids ꢁ Chain elongation ꢁ Bromide
coupling ꢁ Very long chain fatty acids
J Am Oil Chem Soc (2021).
Abbreviations
C32
14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-
dotriacontahexaenoic acid
C34
16(Z),19(Z),22(Z),25(Z),28(Z),31(Z)-
tetratriacontahexaenoic acid
C36
18(Z),21(Z),24(Z),27(Z),30(Z),33(Z)-
hexatriacontahexaenoic acid
20(Z),23(Z),26(Z),29(Z),32(Z),35(Z)-
octatriacontahexaenoic acid
C38
i.e.,
[2,2,3,3,4,4-2H6]14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-
C40
22(Z),25(Z),28(Z),31(Z),34(Z),37(Z)-tetra-
contahexaenoic acid
4(Z),7(Z),10(Z),13(Z),16(Z),19(Z)-
docosahexaenoic acid
22-bromo-3(Z),6(Z),9(Z),12(Z),15(Z),18
(Z)-docosahexaene
N,N-dimethylformamide
dotriacontahexaenoic acid. Also prepared were the monoun-
saturated long chain fatty acids 15(Z)-octadecenoic acid and
15(Z)-tetracosenoic acid. Very long chain fatty acids have
been isolated from retina and other tissues and are of biologi-
cal relevance. The methodology described will assist in further
analytical and biological studies in this field.
DHA
DHA-Br
DMF
NiCl2(glyme) nickel(II) chloride ethyleneglycol dimethyl
ether complex
terpyridine
* Mats Hamberg
4,40,400-tri-tert-butyl-2,20:60,200-terpyridine
mats.hamberg@ki.se
1
Department of Medical Biochemistry and Biophysics and
Larodan Research Laboratory, Karolinska Institutet, Stockholm,
S-171 77, Sweden
Introduction
This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
Elongation of fatty acid acyl chains is a common task in
lipid synthetic chemistry. Apart from producing higher
homologues, such procedures can be used for the incorpora-
tion of isotopic label into specific positions. For practical
J Am Oil Chem Soc (2021)

J Am Oil Chem Soc
work, several methods are available and include for one-
carbon elongation the Arndt-Eistert synthesis, whereby the
fatty acid is converted into a diazoketone which is
rearranged into the elongated ester (Wotiz and Buco, 1955).
An alternative procedure for adding one carbon consists of
transforming the fatty acid into its corresponding bromide,
which can be further converted into the nitrile and subse-
quently hydrolyzed to provide the elongated product. Two-
carbon elongation is classically performed by malonic ester
synthesis (Klenk and Mohrhauer, 1960; Spener and
Mangold, 1973). Although this is a widely used procedure
it has drawbacks when applied to polyunsaturated fatty
acids since it involves exposure to strong base and high
temperatures. An improved method for C2-elongation
Bromoesters
Methyl esters of ω-bromoacids of chain lengths 2–7,
10, 12, and 16 were obtained from Sigma-Aldrich
or TCI.
Methyl [2,2,3,3,4,4-2H6]10-bromodecanoate was pre-
pared by anodic coupling (Hamberg, 1971) of deuterated
methyl hydrogen glutarate to 7-bromoheptanoic acid
(Scheme 1). [2,2,3,3,4,4-2H6]glutaric anhydride purchased
from C/D/N isotopes (Pointe-Claire, Quebec, Canada) (1 g, 8.3
mmol) was added to 0.37 g (11.6 mmol) of dry methanol and
the mixture stirred at 23ꢀC for 15 hours and then kept at 50ꢀC
for 1 hours. The deuterated methyl hydrogen glutarate thus
formed (about 8.3 mmol) together with 3.1 g (14.8 mmol) of
7-bromoheptanoic acid was added to methanol (80 mL) con-
taining sodium methoxide (1.5 mmol). The solution was elec-
trolyzed for 1.5 hours in a thermostatted cell (about 15ꢀC)
using Pt electrodes and a current of 1 A (Scheme 1).
Extraction with diethyl ether afforded a product from
which the title compound was isolated in 39% yield by
silica gel chromatography followed by purification by
reversed-phase HPLC (column, 250 × 10 mm Nucleosil
C18, solvent system, acetonitrile-water 8:2 [v/v] at a flow
rate of 4 mL min−1). The mass spectrum showed promi-
nent ions at m/z 270/272 (M+), 239/241 (M+–OCH3), 191
(M+–Br), and 77 ([CD2 = C[OD]-OCH3]+). GC–MS
analysis further showed that the sample was >99% deu-
terated and had the following isotopic composition:
89.5% d6, 8.8% d5, 1.7% d4, and less than 0.2% of d3, d2,
d1 and, d0 species. The presence of d5 and d4 species
suggested a limited exchange of deuterium in the car-
boxyl α position, most likely taking place during the
anodic coupling step.
involving
formation
and
fragmentation
of
a
2,2-dimethyloxazoline intermediate has been published
(Kuklev and Smith, 2006). The Kolbe anodic cross-coupling
of fatty acids with shorter-chain half-esters offers a versatile
method for elongation of acyl chains (Schäfer, 1979). Satu-
rated and monounsaturated fatty acids can be used in this
procedure but when applied to polyunsaturated fatty acids
yields are generally poor. Coupling of halides to metal
derivatives of alkyl halides, such as the Li2CuCl4-catalyzed
coupling of Grignard compounds, offers another flexible
method to obtain longer acyl chains (Frisch and
Beller, 2005). A drawback is the need of protection and
deprotection of reactive functional groups such as alcohol,
carboxyl or ester.
The present paper describes a Wurtz type coupling proto-
col for generation of elongated acyl chains. It is based on a
nickel-catalyzed manganese-promoted method for bromide
coupling described by Prinsell et al. (2010). Application to
the bromide derivative prepared from 4(Z),7(Z),10(Z),13
(Z),16(Z),19(Z)-docosahexaenoic acid (DHA) and several
ω-bromoesters as coupling partners resulted in a series of
very long chain fatty acids previously isolated from vertebrate
retina (Aveldano, 1987; Aveldano and Sprecher, 1987) and
later found to serve as precursors of biologically active oxy-
genated derivatives, socalled elovanoids (Bazan, 2018).
Methyl 14-bromotetradecanoate was synthesized in a yield
of 38% by anodic coupling of methyl hydrogen azelate
(20 mmol) to 7-bromoheptanoic acid (10 mmol) as described
above. The pure bromoester was obtained following
purification by silica gel chromatography and reversed-phase
high-performance
chromatography
(solvent
system,
acetonitrile-water 9:1 [v/v]). Methyl 18-bromooctadecanoate
was prepared in an analogous way starting with methyl hydro-
gen succinate and 16-bromohexadecanoic acid.
Materials and Methods
Alkyl Bromides
DHA was purchased from NuChek, Elysian, MN, USA.
15(Z)-octadecenoic acid as well as 15(Z)- and 15(E)-
tetracosenoic acids were products of Larodan Co., Stockholm,
Sweden. Nickel(II) chloride ethyleneglycol dimethyl
ether complex (NiCl2(glyme)), 4,40,400-tri-tert-butyl-2,20:60,200-
terpyridine (terpyridine), manganese, and anhydrous N,N-
dimethylformamide (DMF) were purchased from Sigma-
Aldrich Sweden AB, Stockholm, Sweden.
22-Bromo-3(Z),6(Z),9(Z),12(Z),15(Z),18(Z)-docosahexaene
(DHA Br) was prepared in 90% yield by LiAlH4 reduction
of DHA (1 g) followed by treatment with triphenylphosphine
dibromide in dry dichloromethane and purification by silica
gel chromatography (Scheme 2). In the same way, 1-bromo-9
(Z)-octadecene was prepared from methyl oleate. 8-Bromo-3
(Z)-octene was obtained by bromination of 5(Z)-octen-1-ol
purchased from Sigma-Aldrich.
J Am Oil Chem Soc (2021)

J Am Oil Chem Soc
D
D
Anodic coupling with
HOOC-(CH2)6-Br
D
D
O
D
D
D
D
D
D
D
D
CH3OH
D
D
H3COOC
Br
H3COOC
COOH
O
D
D
D
D
O
1) NiCl2(glyme),
terpyridine, Mn, DHA-Br
2) OH-
D
D
D
D
COOH
D
D
Scheme 1 Synthesis of methyl [2,2,3,3,4,4-2H6]10-bromodecanoate
and [2,2,3,3,4,4-2H6]14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-
dotriacontahexaenoic acid (d6-C32)
1) LiAlH4
2) Phe3PBr2
CH2Br
COOH
DHA-Br
DHA
1) NiCl2(glyme), terpyridine, Mn
Br
COOCH3
2) OH-
α
COOH
ω
C36
Scheme 2 Synthesis of 18(Z),21(Z),24(Z),27(Z),30(Z),33(Z)-hexatriacontahexaenoic acid (C36) by coupling of 22-bromo-3(Z),6(Z),9(Z),12
(Z),15(Z),18(Z)-docosahexaene (DHA Br) to methyl 14-bromotetradecanoate. The points of cleavage generating the α- and ω-ions in the mass
spectrum of the methyl ester of C36 are indicated
Gas–Liquid Chromatography—Mass Spectrometry
(GC–MS)
temperature of 320ꢀC and kept at this temperature for
15 min.
GC–MS was performed with an Agilent 5977E series
GC/MSD equipped with a capillary column of 5%
phenylmethylsiloxane (12 m × 0.2 mm, film thickness
0.33 μm) using helium as the carrier gas. For most runs, the
oven temperature was programmed from 50ꢀC at a rate of
20ꢀC min−1 or from 80ꢀC at a rate of 10ꢀC min−1 to a final
Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out with
DC-Fertigplatten Kieselgel 60 from Merck and a solvent
system of hexane-ethyl acetate-acetic acid 90:10:0.5 (v/v/
J Am Oil Chem Soc (2021)

J Am Oil Chem Soc
v). Spots were visualized by spraying with pho-
sphomolybdic acid and heating at 120ꢀC.
methyl 15-octadecenoate, and dimethyl eicosane-1,-
20-dioate in a ratio of 1:6.5:9. Silica gel chromatography
afforded methyl 15(Z)-octadecenoate (0.22 mmol; purity
>98%). The possible presence of the 15(E) isomer in this
material was examined by GC–MS using the authentic
materials as references. Although <0.3% of the E isomer
was detectable, this level was also observed for the starting
8-bromo-3(Z)-octene. Saponification of the methyl 15(Z)-
octadecenoate and crystallization from hexane at -20ꢀC
gave pure 15(Z)-octadecenoic acid.
Methyl 15(Z)-tetracosenoate was prepared using the
standard protocol starting with 1-bromo-9(Z)-octadecene
and methyl 6-bromohexanoate. Following purification by
silica gel chromatography, a sample was analyzed by GC–
MS using authentic methyl 15(Z)- and 15(E)-
tetracosenoates as references. The methyl tetracosenoate
synthesized was >98% of the 15(Z) isomer but contained a
trace (<1.4%) of the 15(E) isomer. 15(Z)-Tetracosenoic
acid was obtained following saponification and crystalliza-
tion from hexane.
NMR Spectroscopy
1H NMR spectra were recorded using a Bruker 500 MHz
instrument. The solvent was deuteriochloroform and tetra-
methylsilane was used as internal chemical shift standard.
Procedure for Preparation of Very Long Chain Fatty
Acids
For synthesis of very long chain fatty acids having chain
lengths of 32–40 carbons (fatty acids C32–C40), DHA-Br
(113 mg, 0.3 mmol) and methyl ω-bromoalkanoate
(0.9 mmol) were dissolved in 2 mL of dry DMF and
NiCl2(glyme) (8 mg, 36 μmol), terpyridine (13 mg, 32
μmol), and manganese powder (73 mg, 1.3 mmol) were
added. The suspension was mixed, purged with argon, and
stirred at 40ꢀC for 4 hours. The product isolated by extrac-
tion with diethyl ether was subjected to silica gel chroma-
tography (elution with diethyl ether-hexane 3:97 [v/v]) to
provide the elongated ester (yields, 70–75% based on
DHA-Br added, purity >90%). Subsequent treatment with
0.3 M NaOH in 85% ethanol under argon at 60ꢀC for
1 hours afforded the elongated acid, which was further
purified on a second silica gel column (elution with diethyl
ether-hexane-acetic acid 10:90:0.05 (v/v/v); fractions moni-
tored either by TLC or by GC–MS after methylation). In
this way, were obtained C32, C34, C36, C38, C40 as well
as [2,2,3,3,4,4-2H6]C32 having >98% purity as judged by
TLC and by GC–MS analysis of their methyl ester
derivatives.
14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-
Dotriacontahexaenoic Acid (C32)
A colorless oil. The mass spectrum of the methyl ester
derivative showed prominent ions at m/z 482 (M+), 451
(M+–OCH3), 413 (M+–CH2–CH=CH–CH2–CH3), 306
(M+ − [C13H19 + H], “α-ion”), 133, 108 ([C8H12]+,
“ω-ion”), and 79. The sample was homogeneous on TLC
(Rf = 0.17).
16(Z),19(Z),22(Z),25(Z),28(Z),31(Z)-
Tetratriacontahexaenoic Acid (C34)
Synthesis of 15(Z)-octadecenoic acid was carried out
using the above protocol starting with 8-bromo-3(Z)-octene
(0.3 mmol) and methyl 10-bromodecanoate (0.9 mmol).
GC–MS analysis showed that 96% of the material recov-
ered was due to the coupling products 3,13-hexadecadiene,
A colorless viscous oil which solidified at +4ꢀC. The mass
spectrum of the methyl ester derivative showed prominent
ions at m/z 510 (M+), 479 (M+–OCH3), 441 (M+–CH2–
CH=CH–CH2–CH3), 334 (M+ – [C13H19 + H], “α-ion”),
133, 131, 108 ([C8H12]+, “ω-ion”), and 79. The sample
was homogeneous on TLC (Rf = 0.18).
Table 1 1H NMR spectral data of 18(Z),21(Z),24(Z),27(Z),30(Z),33
(Z)-hexatriacontanoic acid (C36)
18(Z),21(Z),24(Z),27(Z),30(Z),33(Z)-
Hexatriacontahexaenoic Acid (C36)
Proton
Chemical shift,
multiplicity (J, Hz)
H-2
2.32–2.37 t (7.5)
1.59–1.67 quint (7.3)
1.22–1.37 m
2.02–2.12 quint (7.5)
5.30–5.42 m
A semisolid which could be recrystallized from methanol at
+4ꢀC; melted to a colorless oil at about 35ꢀC. The mass
spectrum of the methyl ester derivative showed prominent
ions at m/z 538 (M+), 507 (M+–OCH3), 469 (M+–CH2–
CH=CH–CH2–CH3), 362 (M+ − [C13H19 + H], “α-ion”),
H-3
H-4 to H-16
H-17, H-35
H-18, H-19, H-21, H-22, H-24, H-25, H-27,
H-28, H-30, H-31, H-33, H-34
1
133, 131, 108 ([C8H12]+, “ω-ion”), and 79. H NMR spec-
H-20, H-23, H-26, H-29, H-32
H-36
2.79–2.87 m
0.97 t (7.4)
tral data are given in Table 1. The sample was homoge-
neous on TLC (Rf = 0.20).
J Am Oil Chem Soc (2021)

J Am Oil Chem Soc
20(Z),23(Z),26(Z),29(Z),32(Z),35(Z)-
Octatriacontahexaenoic Acid (C38)
83, 69, and 55. The gas chromatogram showed a trace
(<1.4%) of the later eluting 15(E) isomer. 15(Z)-
Tetracosenoic acid was a crystalline solid and proved iden-
tical to an authentic sample.
A solid, which could be recrystallized from methanol at +4ꢀC
affording material having m.p. 40.5–41.0ꢀC. The mass spec-
trum of the methyl ester derivative showed prominent ions at
m/z 566 (M+), 535 (M+ − OCH3), 497 (M+ − CH2–
CH=CH–CH2–CH3), 390 (M+ − [C13H19 + H], “α-ion”),
131, 108 ([C8 H12]+, “ω-ion”), and 79. The sample was
homogeneous on TLC (Rf = 0.22).
Results and Discussion
Coupling of alkyl bromides in the presence of metallic
sodium in diethyl ether was originally described by
Adolphe Wurtz (Wurtz, 1855), however, use of this method
for reductive dimerization is hampered by variable yields
and limited tolerance to functional groups. Modifications
using metals such as Cu, Fe, and Pd have been developed
but many of them appear to have drawbacks for the present
purpose.
Recently Weix and coworkers elaborated a method for
nickel-catalyzed dimerization of alkyl halides, alkyl pse-
udohalides, and allylic acetates (Prinsell et al., 2010). The
procedure uses 1 eq. of metallic Mn and catalytic amounts
of NiCl2(glyme) and terpyridine and is performed in DMF
at moderate temperature. The mildness of the method and
the lack of involvement of strong acid or base made it
attractive for use in the polyunsaturated fatty acid field.
Although described for dimerization of single components
(Prinsell et al., 2010), initial experimentation in our labora-
tory revealed that mixed coupling of two different bro-
22(Z),25(Z),28(Z),31(Z),34(Z),37(Z)-
Tetracontahexaenoic Acid (C40)
A solid which was recrystallized from methanol at +4ꢀC
affording material having m.p. 48.0–48.5ꢀC. The mass
spectrum of the methyl ester derivative showed prominent
ions at m/z 594 (M+), 563 (M+ − OCH3), 525 (M+ − CH2–
CH=CH–CH2–CH3), 418 (M+ − [C13H19 + H], “α-ion”),
131, 108 ([C8 H12]+, “ω-ion”), and 79. The sample was
homogeneous on TLC (Rf = 0.25).
[2,2,3,3,4,4-2H6]14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-
Dotriacontahexaenoic Acid (d6-C32)
A colorless oil. The mass spectrum of the methyl ester
derivative showed prominent ions at m/z 488 (M+), 457
(M+ − OCH3), and 419 (M+ − CH2-CH=CH-CH2-CH3).
As expected, the α-ion appearing at m/z 306 in the
unlabeled molecule was shifted to m/z 312 whereas the
ω-ion remained at m/z 108. The specimen was due to >99%
deuterated molecules and had an isotopic composition of
84.0% hexadeuterated (d6) molecules, 14.0% d5, 2.0% d4,
and less than 0.2% of d3, d2, d1, and d0 species.
mides, i.e.,
a
long-chain alkyl bromide and an
ω-bromoester, resulted in good yields of elongated ester
provided that an excess of bromoester (3:1) was used. The
product consisted of a hydrocarbon, a monoester and a
diester. These were readily separated by silica gel chroma-
tography, affording the desired monoester in a purity of
>90%. The yields based on alkyl bromide used (0.3 mmol)
were 70–75%. Saponification and purification by silica gel
chromatography yielded the corresponding free acid in a
purity of >98%.
Methyl 15(Z)-Octadecenoate and 15(Z)-
Octadecenoic Acid
The methyl ester was a colorless oil whose mass spectrum
showed prominent ions at m/z 296 (M+), 264
(M+ − CH3OH), 222, 180, 152, 74, and 55. The gas chro-
matogram showed a trace (<0.3%) of the later eluting
15(E) isomer. 15(Z)-Octadecenoic acid was a crystalline
solid, m.p. 41.5–42.0ꢀC (lit. 41–42ꢀC, (Rawling
et al., 2010). Its analytical data were identical to those of an
authentic sample.
The Wurtz metal-promoted coupling proceeds by single-
electron transfer and an alkyl radical, and it therefore
seemed important to check for possible radical-induced
cis-trans isomerization of double bonds. NMR analysis of
the very long chain fatty acids synthesized gave no
indication for the presence of trans double bond(s), how-
ever, the complexity of the signals from the six double
bonds at 5.30–5.42 p.p.m. (Table 1) necessitated more
precise tests. First, methyl 15-octadecenoate was synthe-
sized by coupling of 8-bromo-3(Z)-octene to methyl
10-bromodecanoate. Analysis by GC–MS under conditions
where the Z and E isomers of methyl 15-octadecenoate sep-
arate revealed that the product was >99.7% of the 15(Z)
isomer. Second, 1-bromo-9(Z)-octadecene was coupled to
methyl 6-bromohexanoate. The methyl 15-tetracosenoate
Methyl 15(Z)-Tetracosenoate and 15(Z)-
Tetracosenoic Acid
The methyl ester was a colorless oil whose mass spectrum
was identical to that of an authentic sample and showed
ions at m/z 380 (M+), 348 (M+ − CH3OH), 306, 264,
J Am Oil Chem Soc (2021)

J Am Oil Chem Soc
Acknowledgment The financial support from Sune Bergströms
fond at the Karolinska Institutet is gratefully acknowledged.
thus formed was analyzed by GC–MS using authentic
methyl 15(Z)- and 15(E)-tetracosenoates as references. The
tetracosenoate synthesized was >98% of the 15(Z) isomer
although a small peak (<1.4%) due to the 15(E) isomer was
also observed. From these experiments it was concluded
that no or only insignificant (Z) - > (E) isomerization of
double bonds took place during the coupling reaction. In
cases, where a > 99% pure product is needed, further puri-
fication by RP-HPLC or crystallization may be required.
As targets for the coupling method were chosen the very
long chain fatty acids isolated from bovine retina and other
tissues (Aveldano, 1987; Aveldano and Sprecher, 1987;
Bazan, 2018). DHA Br, the bromo derivative of doco-
sahexaenoic acid prepared by reduction of DHA to the alco-
hol followed by bromination, was coupled to ω-bromoesters
having chain lengths of 10–18 carbon atoms. In this way, a
set of very long chain (32–40 carbons) fatty acids could be
prepared in good yield (Scheme 2). Such compounds have
previously been obtained using multistep syntheses involving
Li2CuCl4-catalyzed coupling of Grignard reagents (Raman
and Tarwade, 2013). The deuterated very long chain fatty acid
prepared, i.e., [2,2,3,3,4,4-2H6]14(Z),17(Z),20(Z),23(Z),26
(Z),29(Z)-dotriacontahexaenoic acid (Scheme 1), appears to
be the first example of a very long chain fatty acid obtained in
isotopically labeled form.
Conflict of Interest The authors declare that they have no conflict
of interest.
References
Aveldano, M. I. (1987) A novel group of very long chain polyenoic
fatty acids in dipolyunsaturated phosphatidylcholines from verte-
brate retina. Journal of Biological Chemistry, 262:1172–1179.
Aveldano, M. I., & Sprecher, H. (1987) Very long chain (C24 to C36
)
polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated
phosphatidylcholines from bovine retina. Journal of Biological
Chemistry, 262:1180–1186.
Bazan, N. G. (2018) Docosanoids and elovanoids from omega-3 fatty
acids are pro-homeostatic modulators of inflammatory responses, cell
damage and neuroprotection. Molecular Aspects of Medicine, 64:18–33.
Brauner, A., Budzikiewicz, H., & Boland, W. (1982) Studies in chem-
ical ionization mass-spectrometry. Organic Mass Spectrometry, 17:
161–164.
Frisch, A. C., & Beller, M. (2005) Catalysts for cross-coupling reac-
tions with non-activated alkyl halides. Angewandte Chemie Inter-
national Edition, 44:674–688.
1
Gunstone, F. D. (1990) H- and 13C-NMR spectra of six n-3 polyene
esters. Chemistry and Physics of Lipids, 56:227–229.
Hamberg, M. (1971) Steric analysis of hydroperoxides formed by
lipoxygenase oxygenation of linoleic acid. Analytical Biochemistry,
43:515–526.
Klenk, E., & Mohrhauer, H. (1960) Untersuchungen über den
Stoffwechsel der Polyenfettsäuren bei der Ratte. Hoppe-Seyler’s
Zeitschrift für Physiologische Chem, 320:218–232.
As expected, the methyl esters of the long chain acids
synthesized on GC gave a homologous series of peaks
showing considerable broadening (Aveldano, 1987;
Aveldano and Sprecher, 1987). Their identities were
supported by the mass spectra, which all showed the
socalled “ω-ion” at m/z 108 ([C8H12]+) typical of ω3-fatty
acid esters (Mjøs, 2004). Another distinctive ion was the
socalled “α-ion” (M+ − [C13H19 + H]) formed by cleav-
age at the methylene group subsequent to the two first
double bonds counted from the carboxyl end
(e.g., cleavage at C-23/C-24 in case of acid C36;
Scheme 2) (Brauner et al., 1982; Mjøs, 2004). The 1H
NMR spectrum recorded on C36 further supported the
structure by showing signals typical for DHA
(Gunstone, 1990; Sandri and Viala, 1995) as well as a
strong signal at 1.22–1.37 p.p.m. due to the hydrogens at
carbons C-4 to C-16. DHA has a melting point of -44ꢀC.
Although C32 and C34 were also oils at room tempera-
ture, C36 was a semisolid and C38 and C40 were crystal-
line solids having m.p. 40.5–41.0ꢀC and 48.0–48.5ꢀC,
respectively.
Kuklev, D. V., & Smith, W. L. (2006) Chemical C2-elongation of polyun-
saturated fatty acids. Chemistry and Physics of Lipids, 144:172–177.
Mjøs, S. A. (2004) The prediction of fatty acid structure from selected
ions in electron impact mass spectra of fatty acid methyl esters.
European Journal of Lipid Science and Technology, 106:550–560.
Prinsell, M. R., Everson, D. A., & Weix, D. J. (2010) Nickel-cata-
lyzed, sodium iodide-promoted reductive dimerization of alkyl
halides, alkyl pseudohalides, and allylic acetates. Chemical Com-
munications, 46:5743–5745.
Raman, K. & Tarwade, V. (2013) Synthesis and use of omega-3 and
omega-6 very long chain polyunsaturated fatty acids (VLC-PUFA).
United States Patent Application Publication, 2013/0190399 A1.
Rawling, T., Duke, C. C., Cui, P. H., & Murray, M. (2010) Facile and
stereoselective synthesis of (Z)-15-octadecenoic acid and (Z)-
16-octadecenoic acid: Monounsturated omega-3 fatty acids. Lipids,
45:159–165.
Sandri, J.,
& Viala, J. (1995) Syntheses of all-(Z)-5,8,11,14,
17-eicosapentaenoic acid and all-(Z)-4,7,10,13,16,19-docosahexaenoic
acid from (Z)-1,1,6,6-tetraisopropoxy-2-hexene. Journal of Organic
Chemistry, 60:6627–6630.
Schäfer, H. J. (1979) Recent synthetic applications of the Kolbe elec-
trolysis. Chemistry and Physics of Lipids, 24:321–333.
Spener, F., & Mangold, H. K. (1973) Reactions of aliphatic meth-
anesulfonates VII. Chain elongation by two methylene groups.
Chemistry and Physics of Lipids, 11:215–218.
Wotiz, J. H., & Buco, S. N. (1955) The Arndt-Eistert synthesis of
unsaturated acids. Journal of Organic Chemistry, 20:210–214.
Wurtz, A. (1855) Über eine neue Klasse organsiche Radikale.
Annalen der Chemie und Pharmacie, 96:364–375.
The scope of the present coupling method is under fur-
ther study. Of particular interest is its possible use in larger
scale syntheses as an alternative to anodic coupling and
other coupling procedures currently used in the fatty
acid field.
J Am Oil Chem Soc (2021)