Regio- and stereospecific syntheses and nitric oxide donor properties of (E)-9- and (E)-10-nitrooctadec-9-enoic acids

Article abstract of DOI:10.1021/ol060548w

Nitrated fatty acids act as endogenous peroxisome proliferator-activated receptor γ (PPARγ) ligands and nitric oxide (NO) donors. We describe the first specific preparation of the two regioisomers of nitrooleic acid, (E)-9-nitrooctadec-9-enoic acid (1) an

Full text of DOI:10.1021/ol060548w

ORGANIC
LETTERS
2006
Vol. 8, No. 11
2305-2308
Regio- and Stereospecific Syntheses
and Nitric Oxide Donor Properties of
(E)-9- and (E)-10-Nitrooctadec-9-enoic
Acids
Michael J. Gorczynski, Jinming Huang, and S. Bruce King*
Department of Chemistry, Wake Forest UniVersity,
Winston-Salem, North Carolina 27109
kingsb@wfu.edu
Received March 6, 2006
ABSTRACT
Nitrated fatty acids act as endogenous peroxisome proliferator-activated receptor
γ
(PPARγ) ligands and nitric oxide (NO) donors. We describe
the first specific preparation of the two regioisomers of nitrooleic acid, (E)-9-nitrooctadec-9-enoic acid (1) and (E)-10-nitrooctadec-9-enoic acid
(2), from cis-cyclooctene and monomethyl azelate, respectively. These syntheses rely upon a Henry condensation between a nine-carbon nitro
component and a nine-carbon aldehyde. Preliminary chemiluminescence NO detection studies reveal the ability of these nitrated fatty acids
to release NO.
Nitrated fatty acids have emerged as a unique class of
endogenously produced signaling molecules. Initial studies
reveal that nitrated derivatives of linoleic acid (18:2) occur
in concentrations of ∼500 nM in human red blood cells and
plasma, making nitrated fatty acids the single largest pool
of bioactive nitrogen oxides in the vasculature.1 Additional
studies show that nitrolinoleate (LNO2) acts as a ligand of
the peroxisome proliferator-activated receptor γ (PPARγ).2
Activation of this transcription factor results in gene expres-
sion that affects numerous critical cellular processes including
growth and differentiation, inflammation, metabolic homeo-
stasis, and vasomotor processes.3 Other studies show that
LNO2 also spontaneously releases nitric oxide (NO), an
endogenous free-radical signaling mediator that regulates a
number of biological processes including blood pressure,
neurotransmission, and platelet aggregation.4,5 These interest-
ing molecules thus bridge fatty acid-derived signaling with
(1) (a) Lima, E. S.; Di Mascio, P.; Rubbo, H.; Abdalla, D. S. P.
Biochemistry 2002, 41, 10717. (b) Baker, P. R. S.; Schopfer, F. J.; Sweeney,
S.; Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11577.
(2) Schopfer, F. J.; Lin, Y.; Baker, P. R. S.; Cui, T.; Garcia-Barrio, M.;
Zhang, J.; Chen, K.; Chen, Y. E.; Freeman, B. A. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 2340.
(3) (a) Bishop-Bailey, D.; Wray, J. Prostaglandins Other Lipid Mediat.,
2003, 71, 1. (b) Bishop-Bailey, D. Br. J. Pharmacol, 2000, 129, 823. (c)
Dussault, I.; Forman, B. M. Prostaglandins Other Lipid Mediat. 2000, 62,
1. (d) Hihi, A. K.; Michalik, L.; Wahli, W. Cell. Mol. Life Sci. 2002, 59,
790. (e) Na, H. K.; Surh, Y. J. Biochem. Pharmacol. 2003, 66, 1381.
10.1021/ol060548w CCC: $33.50
© 2006 American Chemical Society
Published on Web 04/25/2006

Scheme 1. Retrosynthetic Analysis for Nitrooleic Acid Isomers 1 and 2
nitric oxide-mediated signaling. More recent work shows that
reaction. Elimination of water from the corresponding nitro
alcohols followed by hydrolysis would form the nitroalkenes
1 and 2.
(E)-9- and (E)-10-nitrooctadec-9-enoic acids (nitrated oleic
acids, OA-NO2) occur with a higher prevalence in human
blood and urine than LNO2.6 Additionally, OA-NO2 activates
PPARγ with a greater potency than LNO2.6
Current methods for the synthesis of these nitrated fatty
acids involve the modification of previous nitro-selenylation
strategies used to synthesize conjugated nitroalkenes.7,8
Alternatively, addition of nitronium tetrafluoroborate to an
unsaturated fatty acid or fatty acid hydroperoxide also forms
nitrated fatty acids.9 These nonspecific procedures form
regioisomeric mixtures of nitroalkene products that require
multiple purification steps. More importantly, these nonspe-
cific syntheses prevent strict analysis of the structural
requirements for the biological activity of these unique
molecules. Here we report the first regio- and stereospecific
syntheses of (E)-9-nitrooctadec-9-enoic acid (1) and (E)-10-
nitrooctadec-9-enoic acid (2) and show that these nitrated
lipids spontaneously release nitric oxide.
Scheme 2. Synthesis of (E)-9-Nitrooctadec-9-enoic Acid, 1
Scheme 1 describes the retrosynthetic strategy for prepara-
tion of the specific nitrooleic acid regioisomers. The basic
strategy relies upon the nitro aldol condensation (Henry
reaction) between a nine-carbon nitro component and nine-
carbon aldehyde as the critical carbon-carbon bond-forming
(4) (a) Lima, E. S.; Bonini, M. G.; Augusto, O.; Barbeiro, H. V.; Souza,
H. P.; Abdalla, D. S. P. Free Radic. Biol. Med. 2005, 39, 532. (b) Schopfer,
F. J.; Baker, P. R. S.; Giles, G.; Chumley, P.; Batthyany, C.; Crawford, J.;
Patel, R. P.; Hogg, N.; Branchaud, B. P.; Lancaster, Jr., J. R.; Freeman, B.
A. J. Biol. Chem. 2005, 280, 19289.
(5) (a) Kerwin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L. J. Med.
Chem. 1995, 38, 4343. (b) Umans, J. G.; Levi, R. Annu. ReV. Physiol. 1995,
57, 771. (c) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.;
Janczuk, A. J. Chem. ReV. 2002, 102, 1091 and references therein. (d)
Thatcher, G. R. J. Curr. Top. Med. Chem. 2005, 5, 597.
(6) Baker, P. R. S.; Lin, Y.; Schopfer, F. J.; Woodcock, S. R.; Groeger,
A. L.; Batthyany, C.; Sweeney, S.; Long, M. H.; Iles, K. E.; Baker, L. M.
S.; Branchaud, B. P.; Chen, Y. E.; Freeman, B. A. J. Biol. Chem. 2005,
280, 42464.
(7) Lim, D. G.; Sweeney, S.; Bloodsworth, A.; White, C. R.; Chumley,
P. H.; Krishna, N. R.; Schopfer, F. J.; O’Donnell, V. B.; Eiserich, J. P.;
Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15941.
(8) (a) Hamaya, T.; Tomoda, S.; Takeuchi, Y.; Nomura, Y. Tetrahedron
Lett. 1982, 23, 4733. (b) Seebach, D.; Calderari, G.; Knochel, P. Tetrahedron
1985, 41, 4861.
The regio- and stereospecific synthesis of 1 (Scheme 2)
begins with ozonolysis of cis-cyclooctene to form aldehyde
(3) as previously reported.10 Condensation of 3 with ni-
tromethane and a catalytic amount of potassium tert-butoxide
(t-BuOK) affords nitro alcohol (4).11 Acetylation of 4
(9) (a) O’Donnell, V. B.; Eiserich, J. P.; Bloodsworth, A.; Chumley, P.
H.; Kirk, M.; Barnes, S.; Darley-Usmar, V. M.; Freeman, B. A. Methods
Enzymol. 1999, 301, 454. (b) Napolitano, A.; Camera, E.; Picardo, M.;
d’Ischia, M. J. Org. Chem. 2000, 65, 4853.
(10) Li, G.-Y.; Che, C.-M. Org. Lett. 2004, 6, 1621.
(11) Denmark, S. E.; Kesler, B. S.; Moon, Y.-C. J. Org. Chem. 1992,
57, 7, 4912.
2306
Org. Lett., Vol. 8, No. 11, 2006

of 7. Ultimately, treatment of 7 with 6 M aq HCl at reflux for
12 h forms 1 (δ 7.08 ppm for nitroalkene proton) in 58% yield
(69% yield brsm). Both 1H and 13C NMR spectroscopy con-
firm the presence of a single regioisomer, and chemical shift
analysis again indicates the formation of the E-stereoiosmer.
The synthesis of 2 begins with the borane reduction of
commercially available monomethyl azelate to give alcohol
(8) in 89% yield as previously reported (Scheme 3).14 PCC
oxidation of 8 in CH2Cl2 gives aldehyde (9).14 Preparation
of the nine-carbon nitro-containing component begins with
the condensation of octanal with nitromethane and a catalytic
amound of t-BuOK to give nitro alcohol (10).11 Acetylation
of 10 followed by reduction with a 1 M solution of sodium
borohydride in ethanol affords nitroalkane (11).11,12 Con-
densation of aldehyde (9) and nitroalkane (11) furnishes nitro
alcohol (12) as a mixture of diastereomers in 66% yield.11
Acetylation followed by dehydroacetylation of nitro alcohol
(12) gives 13 as a single regioisomer (δ 7.06 ppm for the
nitroalkene proton).11 Hydrolysis of ester 13 in refluxing 6
N aq HCl for 12 h yields 2 (δ 7.06 ppm for the nitroalkene
proton) in 53% yield (59% yield brsm).
Scheme 3. Synthesis of (E)-10-Nitrooctadec-9-enoic Acid, 2
Chemiluminescence NO detection experiments provide the
first evidence that (E)-9- and (E)-10-nitrooctadec-9-enoic
acids release NO and nitrite (Table 1). Direct analysis of
Table 1. Chemiluminescence Detection of Nitric Oxide and
Nitrite Release from 1, 2, 7, and 13 (50 mM)
a,b
-
a,c
]
solution
incubation
time (h)
[NO]headspace
[NO2
(µM)
compd
(µM)
1
1
3
20
1
3
20
1
1.93 ( 0.11
1.76 ( 0.13
4.23 ( 0.43
2.30 ( 0.02
3.20 ( 0.14
22.09 ( 1.26
3.63 ( 0.45d,e
0e
ND
proceeds smoothly with acetic anhydride using DMAP as a
catalyst and reduction of the corresponding nitro acetates
with a 1 M solution of sodium borohydride in ethanol gives
nitroalkane (5) in 83% yield for the two steps.11,12 Condensa-
tion of nitroalkane (5) with nonanal and a catalytic amount
of t-BuOK gives nitro alcohol (6) as a mixture of diastere-
omers in high yield.11 One-pot acetylation followed by
dehydro-acetylation of 6 exclusively gives the methyl ester
(7) in moderate yield after purification by column chroma-
tography.11 1H NMR chemical shift analysis suggests the
exclusive formation of the E isomer as the alkene proton of
E-nitroalkenes display a characteristic chemical shift of ∼δ
7.00 ppm while the alkene proton of Z-nitroalkenes has a
characteristic chemical shift of approximately δ 5.80 ppm.13
Hydrolysis of the methyl ester of 7 proved somewhat diffi-
cult due to the reactive nature of the nitroalkene. Treatment
of 7 with 1 M aq NaOH (room temperature to 70 °C) fails
to transform 7 to 1 as judged by TLC and 1H NMR. Increas-
ing the temperature to 80 °C or higher resulted in complete
decomposition of 7 (presumably through retro-Henry reac-
tions) with no evidence of 1. Incubation of 7 with iodotrimeth-
ylsilane (TMSI), made in situ from TMSCl and NaI, in re-
fluxing acetonitrile also results in the complete decomposition
10.98 ( 0.77
25.71 ( 0.58
ND
6.63 ( 0.60
23.79 ( 1.27
53.98 ( 4.28f
1.85 ( 0.10f
2
7
13
1
a Dissolved in phosphate buffer (25 mM, pH 7.4). b 500 µL injection.
c 5 µL injection. d Dissolved in 1:1 EtOH/phosphate buffer (25 mM, pH
7.4). e 100 µL injection. f Dissolved in 1:1 EtOH/deionized water. ND )
no data.
the reaction headspace shows the time-dependent formation
of NO upon anaerobic incubation of 1 and 2 in buffer at
room temperature. Analysis of the reaction solution following
KI/acetic acid reduction reveals the time-dependent release
of nitrite from 1 and 2. At this time, it remains unclear
whether nitrite directly forms from the decomposition of 1
and 2 or from further oxidation of NO. Incubation of the
methyl esters (7 and 13) under similar conditions followed
by chemiluminescence NO detection also shows the release
of NO and nitrite (Table 1). While these expeirments clearly
show the spontaneous release of NO from these compounds,
the amount of NO produced is quite low (<1% under these
condidtions) indicating the relative stability of these com-
(12) Wollenberg, R. H.; Miller, S. J. Tetrahedron Lett. 1978, 19, 3219.
(13) (a) Lucet, D.; Heyse, P.; Gissot, A.; Le Gall, T.; Mioskowski, C.
Eur. J. Org. Chem. 2000, 3575. (b) Denmark, S. E.; Gomez, L. J. Org.
Chem. 2003, 68, 8015.
(14) Gung, B. W.; Dickson, H. Org. Lett. 2002, 4, 2517.
Org. Lett., Vol. 8, No. 11, 2006
2307

pounds. It remains to be determined whether this amount of
NO release from these nitrated lipids directly participates in
the observed biological activities of these compounds. These
synthetic studies provide material to further examine the role
of NO in the action of these lipid derivatives.
acids capable of activation of the PPARγ receptor. In
addition, these studies reveal the ability of these compounds
to act as NO donors, similar to nitrated linoleic acid. These
synthetic advances allow a detailed examination of the
relationship between the structure and biology of this newly
discovered class of cell signaling reagents and such studies
are currently underway.
Proposed mechanisms that include nitric oxide release
from nitroalkenes currently include a modified Nef reaction
and a nitroalkene rearrangement to a nitrite ester followed
by N-O bond homolysis.4 Both of these mechanisms yield
carbon-based radicals as organic products, and we have yet
to determine the organic products in our decompositions.
Neither of these mechanisms explains the observed differ-
ences in NO and nitrite release between the regioisomers 1
and 2 and 7 and 13, suggesting possible other operative
pathways for NO release. The developed synthetic pathways
should form the basis of new studies to clearly determine
the rate and mechanism of NO release from these specific
compounds and also whether nitroalkenes represent a new
general class of NO or nitrite donors.4
Acknowledgment. This work was supported by the
National Institutes of Health (HL62198, S.B.K.). The NMR
spectrometer used in this work was purchased with partial
support from the NSF (CHE-9708077) and the North
Carolina Biotechnology Center (9703-IDG-1007).
Supporting Information Available: Full experimental
details for the preparation of all intermediates and products,
as well as complete spectroscopic and analytical data for all
characterized compounds. Copies of 1H and 13C NMR spectra
for 1, 2, 4, 6, 7, 12, and 13 are also included. This material
is available free of charge via the Internet at http://pubs.acs.org.
In summary, this work describes the first regio- and
stereospecific syntheses of (E)-9- and (E)-10-nitrooctadec-
9-enoic acids (1 and 2), endogenously produced nitrated fatty
OL060548W
2308
Org. Lett., Vol. 8, No. 11, 2006