Chemical synthesis of deuterium-labeled and unlabeled very long chain polyunsaturated fatty acids

Article abstract of DOI:10.1016/j.tetlet.2010.09.139

First syntheses of a deuterium-labeled very long C34-containing polyunsaturated fatty acid, 34:5n5, and three other unlabeled very long chain C30-32 containing polyunsaturated fatty acids are reported. These syntheses were achieved by coupling chemically

Full text of DOI:10.1016/j.tetlet.2010.09.139

Tetrahedron Letters 51 (2010) 6426–6428
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Chemical synthesis of deuterium-labeled and unlabeled very long chain
polyunsaturated fatty acids
Ghulam M. Maharvi a, Albert O. Edwards b, , Abdul H. Fauq a,
⇑
⇑
a Chemical Synthesis Core Facility, Mayo Clinic Jacksonville, FL 32246, USA
b Institute for Molecular Biology, University of Oregon, Eugene, OR 97403, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
First syntheses of a deuterium-labeled very long C34-containing polyunsaturated fatty acid, 34:5n5, and
three other unlabeled very long chain C30–32 containing polyunsaturated fatty acids are reported. These
syntheses were achieved by coupling chemically modified C22- and C20-containing polyunsaturated
fatty acids with carbanions derived from arylalkyl sulfones, followed by sodium amalgam-mediated
desulfonylation.
Received 26 August 2010
Revised 23 September 2010
Accepted 27 September 2010
Available online 7 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Very long chain polyunsaturated fatty acids (VLC-PUFAs) are
arbitrarily defined as fatty acids that have 24 or more carbon atoms
in their molecules with cis-double bonds interrupted by methylene
units. These fatty acids found in most living organisms from
humans to autotrophic and heterotrophic lower organisms can
be grouped into two molecular families: omega-6 (or nÀ6) and
omega-3 (or nÀ3) families, where the number 6 or 3 represents
the position of the first double bond proximal to the terminal
methyl group.
Even though aspects of metabolic engineering of some specific
VLC-PUFA are reported,16 to the best of our knowledge, their chem-
ical laboratory synthesis has not been described. In order to evalu-
ate the retinal role of VLC-PUFA in the vision process in our
ongoing epidemiological studies, we have synthesized a variety
of VLC-PUFAs with carbon chains varying between C-30 to C-34
and with three, four, and five cis-oriented C–C double bonds
(Fig. 1). Additionally, we prepared a dideuterated version of a
C-34 omega-3 VLC-PUFA, 34:5n3.d2 to be used as a probe in our
studies. For convenience, all VLC-PUFA materials were synthesized
using C-20 and C-22-containing all-cis fatty acids available com-
mercially (Nu-Chek Prep, Inc). Care was taken to select reagents
that are not expected to invert geometrical integrity of the C–C
double bonds. Additionally, all reactions were carried under argon
in flasks covered with aluminium foil to prevent oxidation.
Our first priority was to prepare a deuterium-labeled omega-3
fatty acid 34:5n3 with a difference of at least 2 mass units. Cost
considerations suggested incorporating two deuterium atoms at
a location near the middle of the saturated part of the long carbon
chain that was less likely to be prone to deuterium exchange under
our bioassay conditions. To this end, 12-bromododecanol (1)
(Scheme 1) was protected with a robust t-butyldiphenylsilyl
(TBDPS) group to give the bromosilyl ether 2. Next, the bromide
function of 2 was displaced with a sulfinate anion in N,N-dimeth-
ylformamide (DMF) to generate the sulfone fragment 3 in 94% yield
over two steps.11
Dietary intervention studies suggest that omega-3 PUFAs such
as eicosapentaenoic acid (EPA; 20:5D5,8,11,14,17) and docosahex-
aenoic acid (DHA; 22:6D4,7,10,13,16,19) confer health benefits to
humans.1–3 PUFAs are also widely distributed in higher plants.4
Humans, like other animals, can synthesize PUFAs from the essen-
tial a
-linolenic acid5 and the PUFAs are distributed throughout the
body. However, the distribution of VLC-PUFAs appears to be more
limited, being found in retina, brain,1 testis, and spermatozoa.6 The
VLC-PUFAs with 3–9 double bonds are not only susceptible to oxi-
dative damage but are also difficult to separate from mammalian
tissues for characterization.7 Therefore, not much is known about
the metabolism and function of VLC-PUFAs.
Animal models of an early on set form for macular degeneration
(autosomal dominant Stargardt-like macular dystrophy, STGD3)
were used to show that this human disease arises due to a defi-
ciency of VLC-PUFAs in the retina.8 Mutation of the gene encoding
elongation of very long chain fatty acid 4 (ELOVL4) leads to STGD3.9
ELOVL4 has been shown to elongate long fatty acids to form VLC-
PUFAs.10 Thus, the VLC-PUFAs present in retina have recently seen
a renewed interest because of their important role in vision.
In a separate pot, omega-3-all-cis-docosapentaenoic acid 4 was
reduced either directly with lithium aluminium deuteride in tetra-
hydrofuran solvent, or via cyanuric fluoride-mediated acyl fluoride
formation12 followed by sodium borodeuteride reduction,13 to give
an alcohol that was immediately converted into the desired deu-
terated p-toluenesulfonate ester 5 in 71% yield over two steps.
For coupling the sulfone fragment 3 with the fatty acid tosylate
fragment 5, the sulfone 3 was deprotonated to a lithocarbanion
⇑
Corresponding authors. Tel.: +1 904 953 8034; fax: +1 904 953 7117 (A.H.F.).
E-mail addresses: edwardsa@uoregon.edu (A.O. Edwards), fauq.abdul@mayo.edu
(A.H. Fauq).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.09.139

G. M. Maharvi et al. / Tetrahedron Letters 51 (2010) 6426–6428
6427
D
D
COOH
10
COOH
8
COOH
3
10
HOOC
10
C5H11
C5H11
C5H11
C2H5
C32:4+1(12)n6
30:3n6
32:4n6
34:5n3.d2
Figure 1. Structures of synthesized VLC-PUFAs.
OH
OTBDPS
10
OTBDPS
imidiazole/DMF,
r.t., 3h, 90%
PhSO2Na
DMF, 70 oC, 3h, 97%
Ph
Cl
Si
Ph
+
10
10
Br
PhO2S
Br
2
1
3
1. LiAlD4/THF, 0 °C to rt, 3h
(96%), or cyanuric
fluoride followed by
NaBD4 reduction
3, n-butyllithium/THF,
-78 to r.t, then 5, 95%
3
D
D
3
COOH
TsO
2. p-TsCl, Py, 0 °C to rt, 74%
5
4
O
R
OH
10
OTBDPS
10
1. (C4H9)4NF/THF, 94%
2. Jones oxidation, 83%
3
3
D
D
D
D
3. 5%Na/Hg, NaH2PO4,
40h, MeOH, 75%
7 (R = SO2Ph);
6
SO2Ph
8 (R = H, C34:5n3.d2)
Scheme 1. Synthesis of 34:5n3.d2.
Ph
O
S O
1. LiAlH4/THF
OTs
OTBDPS
º
3, BuLi,-78ºC,
30 min,
COOH
9a, 9b
THF, 0 C to r.t.,
10
3h, 94%,99%
X
X
X
X
X
X
2. TsCl, Pyridine,
then 12 h,
r.t., 62%, 85%
º
0 C to r.t.,
C5H11
C5H11
C5H11
11a, 11b
O
10a, 10b
4h, 65%, 62%
9a, X = C0H0
9b, X = CH
Ph
O
S O O
OH
10
1. (n-Bu)4NF, THF
r.t., 1h, 99%, 66%
OH
X
X
10%Na/Hg
10
X
X
Na2HPO4/MeOH,
40 h, 84%, 51%
2. Jones reagent
acetone, r.t., 88%, 84%
C5H11
C5H11
13a; C30:3n6 (X = C0H0)
13b; C32:4n6, (X = CH)
12a, 12b
Scheme 2. Synthesis of 32:4n6 and 30:3 n6. (Yields reported in series a, X = C0H0; in series b, X = CH.)
with n-butyllithium in tetrahydrofuran at À78 °C and reacted with
the tosylate 5 to furnish the deuterated sulfone 6 in 95% yield. The
TBDPS group was removed with tetra-n-butylammonium fluoride
(94%) and the resulting alcohol was carefully oxidized with the
Jones reagent14 in acetone (83%) to give the polyunsaturated fatty
acid 7. Finally, the phenylsulfone moiety of 7 was reductively
removed by treating it with sodium amalgam in methanol under
phosphate buffer conditions15 furnishing the VLC-PUFA 34:5n3 8
after purification to homogeneity by flash silica gel chromatogra-
phy. The overall yield of this eight-step convergent synthetic
sequence leading to the VLC-PUFA 8 was 38%.
For some of our ongoing projects, two unlabeled omega-6 VLC-
PUFAs, namely, 30:3n6 and 32:4n6 (Scheme 2) were also synthe-
sized using the methodology described above. These syntheses
employed omega-6-all-cis-octadecatrienoic acid (18:3n6, 9a) and
omega-6-all-cis-icosatetraenoic acid (20:4n6, 9b) as the starting
polyunsaturated fatty acid starting materials. Starting from the sul-
fone 3, the entire synthetic sequence proceeded without incident

6428
G. M. Maharvi et al. / Tetrahedron Letters 51 (2010) 6426–6428
C5H11
C5H11
Swern
Oxidation
LiAlH4
9b
44%
THF, 0 oC
to r.t.,
O
OH
15
14
3h, 99%
OTBDMS
10
1. (n-Bu)4NF
COO
H
THF, r.t., 1h,
88%
C5H11
OTBDMS
Ph3P,
OTBDMS
10
10
KHMDS
Toluene, 25%
10
C5H11
Br
PPh3.Br
17
2. Jones oxid.
Acetone, r.t
83%
Tol
99%
16
19; C32:4+1(12)n6
18
Scheme 3. Synthesis of 32:4+1(12)n6.
and furnished the final VLC-PUFAs 30:3n6 (13a) and 32:4n6 (13b)
with overall yields similar to 8.
Supplementary data
As a negative control, we also synthesized 32:4+1(12)n6, an
unusual VLC-PUFA with a cis C–C double bond at the C-12 position,
separated from the nearest C–C double bond by three methylene
groups rather than the usual one. As graphically represented in
Scheme 3, the omega-6-all-cis-icostetraenoic acid 20:4n6, 9b,
was reduced with lithium aluminium hydride in THF to the pri-
mary alcohol 14, which was then partially oxidized to the aldehyde
15. In a Wittig reaction, the aldehyde 15 was coupled with the
phosphorane derived from phosphonate 17, itself derived from
the protected bromoacohol 16, to give a silyl-protected alcohol
18, from which the alcohol was recovered by desilylation and oxi-
dized under Jones oxidation to the VLC-PUFA 32:4+1(12)n6 19. In
this case, the overall synthetic sequence starting from 9b and
12-bromododecanol (2) proceeded in 8.1% combined yield.
The VLC-PUFAs as well as all the synthetic intermediates were
purified by flash silica gel chromatography and their structures
were confirmed by 1H NMR and ESI/APCI-mass spectroscopy. Fur-
thermore, in view of their expected rapid oxidation, all synthesized
VCL-PUFAs were stored away from light at À80 °C under argon.
They were found to be stable at least for several weeks as judged
by their unchanged spectral properties.
Supplementary data (chemical synthesis procedures and spec-
tral data of the final VLC-PUFAs and their synthetic intermediates)
associated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2010.09.139.
References and notes
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In conclusion, in order to elucidate their biological role in the
vision process and related areas, the first practical and general
chemical synthetic method for several very long chain polyunsatu-
rated fatty acids is described. The overall eight-step synthesis
methodology reported herein proceeded in reasonable yield and
can be employed for most VLC-PUFAs required for activity and role
elucidation in any biological system.
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Acknowledgments
The research was supported by the National Institutes of Health
(EY014467), Bethesda, MD and the Foundation Fighting Blindness,
Owing Mills, MD.