The synthesis of the very long chain polyunsaturated fatty acid (VLC-PUFA) 32:6 n-3

Article abstract of DOI:10.1039/d1ob00491c

This article describes the synthesis of VLC-PUFA 32:6 n-3, D₂-labeled 32:6 n-3, and the uptake of 32:6 n-3 into mouse retinal tissue.

Full text of DOI:10.1039/d1ob00491c

Organic &
Biomolecular Chemistry
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The synthesis of the very long chain
polyunsaturated fatty acid (VLC-PUFA) 32:6 n-3†
Cite this: Org. Biomol. Chem., 2021,
19, 5563
a
Alexander Wade,a Rameshu Rallabandi, a Steven Lucas,a Catrina Oberg,
a
Aruna Gorusupudi, b Paul S. Bernsteinb and Jon D. Rainier
*
Received 13th March 2021,
Accepted 24th May 2021
DOI: 10.1039/d1ob00491c
This article describes the synthesis of VLC-PUFA 32:6 n-3, D2-labeled 32:6 n-3, and the uptake of
32:6 n-3 into mouse retinal tissue.
rsc.li/obc
the presence of lower concentrations of VLC-PUFAs in the
retina.11 It is also worth noting that VLC-PUFA concentrations
Introduction
VLC-PUFAs are fatty acids that contain ≥24 carbon atoms and are lower in patients who suffer from AMD.12 Thus, although
consist of an interesting combination of saturated and unsatu- the evidence is anecdotal at this point, arguments can be
rated hydrocarbon subunits.1 They exist in membranes as made for VLC-PUFAs playing a role in AMD. To date, the lack
phospholipids and are present in very low concentrations in of a ready supply of VLC-PUFAs has kept researchers from
the retina and testes.2–4 Generally VLC-PUFAs do not come gaining a better understanding of their role in both healthy
from dietary sources but are instead generated endogenously eyes and patients suffering from AMD.13 In an effort to over-
from the chain extension of long chain fatty acids, typically come this, we set out to synthesize and study one member of
docosahexaenoic acid (DHA), arachidonic acid (AA), or eicosa- the VLC-PUFA family, C32:6 n-3 (1) along with a deuterium
pentaenoic acid (EPA) by the enzyme ELOVL4 (elongation of labeled analog of 1.14
very long chain fatty acids-4).5 Our interest in these targets not
When we began our efforts, we were aware of two indepen-
only came from their intriguing structures but also from the dent syntheses of our initial target, VLC-PUFA C32:6 n-3.15 One
substantial but still anecdotal evidence that suggests that they synthesis came from Raman and co-workers and involved the
play a role in age-related macular degeneration (AMD) and coupling of a cuprate derived from THP protected 10-bromo-1-
Stargardt’s disease (Fig. 1).
decanol and an alkyl bromide derived from DHA. In a similar
As the name suggests, AMD is a degenerative eye disease fashion to Raman, a synthesis from Maharvi and co-workers
that leads to blurred or loss of vision in the center of the visual involved the coupling of a saturated C-10 sulfone with a poly-
field.6 In light of our aging population, that AMD mostly unsaturated tosylate derived from DHA. Attractive to both
afflicts the elderly makes this disease a significant problem. In approaches was that the generation of the requisite 32 carbon
general, there are two forms of the disease, wet and dry. While chain involved the coupling of two readily available precursors.
therapies have been effective in some patients suffering from As part of our overall plan involved tracking the uptake of syn-
choroidal neovascularization (CNV) a form of wet AMD,7 these thetic VLC-PUFAs from a dietary source to the retina, a
same therapies have not been effective for other forms of problem with both of these approaches had to do with the
AMD.8 Not only are there currently no therapies for dry AMD, challenge of using them to synthesize labeled substrates. In
but the etiology of the disease is not well understood.9 Along spite of this, we did initially explore the Raman synthesis but
these lines, it is interesting that the symptoms associated with were unable to repeat the reported coupling reaction. We did
Stargardt’s disease, a hereditary disease caused by mutations not pursue the Maharvi route both because of our desire to
on the ELOVL4 gene, are similar to those seen in the dry form generate labeled material and because we envisioned that the
of AMD.10 These mutations have been shown to lead to the requisite reductive desulfonation would be limiting. Thus, we
down-regulation of the ELOVL4 enzyme and, as a consequence,
aDepartment of Chemistry, University of Utah, 315 South, 1400 East, Salt Lake City,
UT 84112, USA. E-mail: rainier@chem.utah.edu
bDepartment of Ophthalmology and Visual Sciences, 65 Mario Capecchi Drive,
Moran Eye Center, University of Utah, Salt Lake City, UT 84132, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00491c
Fig. 1 VLC-PUFA 32:6 n-3.
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sought out a modified synthetic strategy to these targets. This for this approach was its relatively low efficiency (8 steps, 6%
work is described here.
overall yield). We attributed the efficiency problem to both the
dimerization problem and the somewhat surprising instability
of the orthoester. In our hands it turned out to be particularly
sensitive subsequent to its coupling with DHA. For example,
the C-32 orthoester containing substrates generally underwent
decomposition when exposed to any acidic conditions includ-
ing SiO2 during chromatography. These issues seemed to be
even more problematic on scale up.
Methods and discussion
Concerns that we had when planning our synthetic approach
to the VLC-PUFA substrates were centered around their suscep-
tibility to oxidation and/or olefin isomerization/migration.
Because of this, we felt that it was necessary to generally avoid
strongly oxidizing, reducing, and acidic conditions. The
approach to 1 that we ultimately identified was related to those
mentioned above with one small but significant difference. We
set out to examine the coupling of a masked decanoic acid
nucleophile with a docosahexaenoic acid (DHA) derived alde-
hyde. Not only might the coupling protocol employing the
aldehyde be more successful and amenable to scale-up but the
resulting 2° alcohol would serve as a precursor to an isotopic
(D) label during the subsequent deoxygenation sequence.
Mindful of these goals and in an attempt to avoid a late-stage
oxidation we generated the coupling precursor 2 representing
the saturated VLC-PUFA subunit from 10-bromodecanoic acid
(Scheme 1).16 DHA derived aldehyde 4 was synthesized from
DHA or the corresponding ester by employing a reduction, oxi-
dation sequence. The addition of the Grignard reagent derived
from orthoester 2 to aldehyde 4 gave the desired 2° alcohol 5
but in low yield and as an inseparable mixture with the
product of homo coupling of the Grignard reagent from 2.
Reduction of the mesylate that came from 5 allowed us to
access orthoester protected VLC-PUFA 6 in a modest 14%
overall yield for the three steps. In contrast to our problems
with the coupling chemistry, the hydrolysis of the orthoester
proved uneventful and provided VLC-PUFA 32:6 n-3 1 in 83%
In light of the difficulties mentioned above, we sought out
an alternate approach to 32:6 n-3 that avoided the orthoester
and settled on THP protected bromodecanol 7 (Scheme 2).
The obvious downside to the use of 7 was that it would
require a late-stage oxidation reaction. Regardless, we were
pleased to find that the C-32 THP protected substrates were
generally better behaved than the orthoesters had been in
our hands. For example, the coupling of the Grignard
reagent from 7 with DHA aldehyde 4 afforded 8 in 73% yield
and was successfully carried out on a multi-gram scale.
Mesylate formation gave 9. Reductive removal of the mesylate
gave 32:6 n-3 alcohol 10 after THP acetal hydrolysis. We were
somewhat surprised when the oxidation of 10 proved to be
challenging. The use of Raman’s protocol and CrO3 and
H5IO6 resulted in unrecognizable mixtures of products.
Reagents like TEMPO or PhI(OAc)2 either resulted in the
decomposition of 10 or the recovery of starting material, pre-
sumably due to solubility issues.17 We eventually found that
a two-step procedure that combined a Swern oxidation with a
subsequent Oxone® oxidation gave VLC-PUFA 32:6n-3 in 46%
yield from 10. Overall, the synthesis of 1 required 6 steps
from 7 and was achieved in 29% overall yield.18,19 To date we
have used this sequence to generate >10 g of 1 for uptake
and visual acuity studies.14
yield over the final two steps. The analytical data (1H NMR, 13
NMR, HRMS, GC-MS) for 1 matched both the reported data
and authentic samples of 1. What diminished our enthusiasm
C
As was stated above, one of the advantages of the coupling
with DHA derived aldehyde 4 was that the resulting alcohol
could be converted into an isotopically labelled VLC-PUFA
whose uptake could be tracked using mass spectrometry.20 To
this goal, we targeted C-11 bis-deuterated VLC-PUFA 14
Scheme 1 1st generation synthesis of VLC-PUFA 32:6 n-3.
5564 | Org. Biomol. Chem., 2021, 19, 5563–5566
Scheme 2 2nd generation synthesis of VLC-PUFA 32:6 n-3.
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(Scheme 3). The treatment of the ketone that comes from the undetectable in the liver, brain, and RBC membranes through-
oxidation of alcohol 8 with LiAlD4 resulted in mono-deuterated out the 12 h time frame. Additional studies in wild type and
alcohol 12. Mesylate formation and a second LiAlD4 reduction EVOVL4 knock-out mice are ongoing.
allowed us to access C-11 bis-deuterated alcohol 13 after THP
acetal hydrolysis. Oxidation per our previously established con-
ditions gave bis-deuterated VLC-PUFA 14. We are currently
examining the properties of 14 including its uptake into the
Conclusions
retina.
In conclusion, described here has been
a synthesis of
In view of the physiological benefits of VLC-PUFAs and the
dysfunction associated with low retinal levels of VLC-PUFAs,
we examined the bioavailability of synthetic 32:6 n-3 delivered
to mice via gavage feeding.21 The mice received a single dose
of 11 mg of 32:6 n-3 and were sacrificed at 0, 6, and 12 h (n =
2 mice per time interval). As shown in Fig. 2, even though peri-
pheral tissue like serum, Red Blood Cells (RBC), and the liver
do not normally contain VLC-PUFAs, in our studies
VLC-PUFAs became detectable in the serum 6 h after oral
administration and reached their highest concentration after
12 h. We were also excited to find a significant increase in 32:6
n-3 in the retina 12 h after feeding. VLC-PUFAs remained
VLC-PUFA 32:6n-3 and a deuterium labelled derivative of this
compound from the coupling of saturated and polyun-
saturated precursors. This approach has the advantage of
being amenable to multi-gram scale-up and that it should be
amenable to the synthesis of other members of the VLC-PUFA
family. In addition to the synthesis, we have studied the bio-
availability of VLC-PUFAs and have observed a rapid increase
in retinal VLC-PUFAs in response to acute supplementation.
This result clearly demonstrates that orally administered
VLC-PUFAs are bioavailable to ocular tissues. In addition to
optimizing the synthesis, studying the properties of VLC-PUFA
32:6 n-3, and examining labelled derivatives, we are currently
examining the synthesis and properties of other members of
the VLC-PUFA family. These efforts will be communicated in
due course.
Author contributions
A.G., P.S.B., and J.D.R. designed research; A.W., R.R., S.L., C.
O., and A.G. performed research. A.W., R.R., S.L., C.O. and J.D.
R.contributed new reagents/analytical tools; all authors ana-
lyzed data; and A.G., P.S.B., and J.D.R. wrote the manuscript.
Conflicts of interest
A. G., R. R., J. D. R., P. S. B., and the University of Utah have
filed a provisional patent application on the chemical syn-
thesis of VLC-PUFAs and their therapeutic effects.
Scheme 3 Synthesis of C-11 bis-deuterium VLC-PUFA 32:6 n-3.
Acknowledgements
The authors are grateful for financial support from the
University of Utah Center on Aging, the Foundation for
Fighting Blindness (TA-NMT-0618-0736-UUT), and
a
Department of Ophthalmology and Visual Sciences core grant
from the NIH (EY14800). A. G. was supported by a Knights
Templar Career starter grant. C. O. was supported from funds
from the University of Utah NSF REU program (1659579). The
authors would also like to thank Dr Hsiaonung Chen and
Dr Peter Flynn for assistance in obtaining mass spectra and
NMR spectra, respectively.
All animal handling procedures used in this study were
approved by appropriate institutional animal care and use
committees and were carried out according to NIH guidelines.
Fig. 2 Bioavailability of VLC-PUFA 32:6 n-3. (a) Serum and retinal
uptake of 32:6 n-3 after single-dose gavage feeding of 11 mg per mouse
(n = 2 mice per time point). N.D. = non-detectable.
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