Synthesis of Site-Specifically Labeled Arachidonic Acids as Mechanistic Probes for Prostaglandin H Synthase

Article abstract of DOI:10.1021/ol0361711

(Matrix presented) Prostaglandin H synthase catalyzes the first committed step in the biosynthesis of prostaglandins and thromboxane. Herein we report the synthesis of four site-specifically labeled arachidonic acids for investigation of the radical inter

Full text of DOI:10.1021/ol0361711

ORGANIC
LETTERS
2004
Vol. 6, No. 3
349-352
Synthesis of Site-Specifically Labeled
Arachidonic Acids as Mechanistic
Probes for Prostaglandin H Synthase
Sheng Peng,† Chris M. McGinley,† and Wilfred A. van der Donk*
Roger Adams Laboratory, Department of Chemistry, UniVersity of Illinois,
600 S. Mathews AVenue, Urbana, Illinois 61801
Vddonk@scs.uiuc.edu
Received November 5, 2003
ABSTRACT
Prostaglandin H synthase catalyzes the first committed step in the biosynthesis of prostaglandins and thromboxane. Herein we report the
synthesis of four site-specifically labeled arachidonic acids for investigation of the radical intermediate formed during this enzymatic reaction.
Two compounds were prepared using a common C9−C11 fragment, while another target was synthesized using a previously reported advanced
intermediate. An alkyne coupling followed by hydrogenation and Wittig reaction was used to prepare the final labeled substrate.
Prostaglandin H synthase, or cyclooxygenase (COX), cata-
lyzes the conversion of arachidonic acid into prostaglandin
H2 in the first committed step in the biosynthesis of pro-
staglandins and thromboxane (Scheme 1).1 These compounds
However, at present, the reaction mechanism for the oxida-
tion of arachidonic acid (AA) has not been fully elucidated.
The first step of the enzymatic reaction has been previously
shown to involve abstraction of a hydrogen from the C-13
position of AA by a tyrosyl radical.2,3 This results in forma-
tion of either a pentadienyl or allyl radical depending on the
conformation of the substrate when bound to the enzyme.
Scheme 1
In previous work we have demonstrated the use of site-
specifically deuterium-labeled substrates to investigate the
structure of the original radical intermediate by using EPR
spectroscopy. These studies showed that in COX-2, the
isozyme linked to inflammation, the initial hydrogen abstrac-
tion leads to formation of a pentadienyl radical intermediate
spanning C11-C15.4 The EPR spectrum shows coupling of
the unpaired electron to six different hydrogens. Using
deuterium labeling, four of these were assigned to protons
(2) (a) Karthein, R.; Dietz, R.; Nastainczyk, W.; Ruf, H. H. Eur. J.
Biochem. 1988, 171, 313-320. (b) Tsai, A.-L.; Kulmacz, R. J. Prostag-
landins Other Lipid Mediators 2000, 62, 231-254.
(3) (a) Tsai, A.-L.; Kulmacz, R. J.; Palmer, G. J. Biol. Chem. 1995, 270,
10503-10508. (b) Tsai, A.-L.; Palmer, G.; Xiao, G. S.; Swinney, D. C.;
Kulmacz, R. J. J. Biol. Chem. 1998, 273, 3888-3894.
have been implicated in numerous processes affecting human
health, including inflammation and cardiovascular disease.
† These authors contributed equally to this work.
(1) (a) Rouzer, C. A.; Marnett, L. J. Chem. ReV. 2003, 103, 2239-2304.
(b) Kulmacz, R. J.; van der Donk, W. A.; Tsai, A. L. Prog. Lipid Res.
2003, 42, 377-404 (c) Smith, W. L.; DeWitt, D. L.; Garavito, R. M. Annu.
ReV. Biochem. 2000, 69, 145-182.
(4) (a) Peng, S.; Okeley, N. M.; Tsai, A.-L.; Wu, G.; Kulmacz, R. J.;
van der Donk, W. A. J. Am. Chem. Soc. 2001, 123, 3609-3610. (b) Peng,
S.; Okeley, N. M.; Tsai, A.-L.; Wu, G.; Kulmacz, R. J.; van der Donk, W.
A. J. Am. Chem. Soc. 2002, 124, 10785-10786.
10.1021/ol0361711 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/14/2004

Scheme 2
Scheme 3
30-80) that are observed with either linoleic acid12 (LA) or
arachidonic acid13 as substrate. Holman and co-workers
showed that the human 15-LOX-1 also displays similarly
large isotope effects with linoleic acid.14 To date, however,
no studies have disclosed kinetic isotope effects of the human
lipoxygenases with their natural substrate, arachidonic acid.
The preparation of arachidonic acids deuterium-labeled at
C10 and C13 reported herein will allow such studies on 8-,
12-, and 15-LOX. We elected to prepare dideuterated
compounds instead of our previous syntheses of stereospe-
cifically mono-deuterium-labeled arachidonic acids4 since it
has been shown with soybean lipoxygenase that the large
primary kinetic isotope effect on abstraction of the pro-S
hydrogen atom leads to a decrease in stereospecificity of the
enzyme.15 That is, with [11S-2H]-LA as substrate, the enzyme
abstracts to some extent the hydrogen atom rather than the
deuterium atom from C11. Obviously, this provides difficul-
ties for assessing the KIE, and hence substrates labeled at
both enantiotopic positions were prepared that will alleviate
these complications.
The preparation of [10,10-2H2]-arachidonic acid and
[11-13C]-arachidonic acid proceeded via the same C9-C11
fragment 2. For the former target, ring opening of â-propi-
olactone by methanol, followed by protection of the ensuing
alcohol with tert-butyldimethylsilyl chloride, yielded methyl
ester 1 (Scheme 3). Quantitative deuterium incorporation at
the R-position was achieved by stirring with Na metal in
MeOD. Selective reduction to the aldehyde 2a using
DIBAL-H afforded the C9-C11 fragment without detectable
loss in deuterium incorporation.
at C11, C13, C15, and one of the protons at C16. The
remaining two strongly coupled hydrogens were proposed
to be located at C10. Similar studies using the COX-1
isozyme produced a signal of unknown structure under
certain conditions,5 which possibly is associated with an allyl
radical. Herein we report the synthesis of two arachidonic
acids site-specifically labeled with 13C at positions 11 and
15 that were designed to test the allyl radical hypothesis.
Furthermore, we prepared [10,10-2H2]-arachidonic acid to
confirm the location of the two remaining strongly coupled
protons in the signal observed in COX-2.
The labeled arachidonic acids described herein are also
useful mechanistic probes for the human lipoxygenases
(LOXs). A number of lipoxygenases in vertebrates are known
that differ in the regioselectivity of oxidation (Scheme 2).6
Their nomenclature is based on the position of hydroper-
oxidation of arachidonic acid.7 5-LOX abstracts an hydrogen
atom from C7 to generate 5-hydroperoxy-5Z,8Z,11Z,13E-
eicosatetraenoic acid (5-HPETE), the precursor to the leuko-
trienes that play key roles in inflammation and broncho-
constriction. The 8- and 12-LOX enzymes abstract a hy-
drogen atom from position 10 to produce hydroperoxides at
positions 8 and 12, respectively. At present, two 15-LOX
isozymes have been identified in humans, 15-LOX-18 and
15-LOX-2.9 The former protein has been implicated to play
a role in atherogenesis.10,11 Both enzymes abstract a hydrogen
atom from C13 and produce 15S-HPETE.
Soybean lipoxygenase, a 15-LOX, has received much
attention for the unusually large kinetic isotope effects (KIE,
The synthesis of the C9-C11 fragment for [11-13C]-
arachidonic acid involved direct displacement of 2-chloro-
ethanol with [13C]-potassium cyanide followed by benzyla-
tion to afford nitrile 3 in good yield (Scheme 3). [13C]-
Potassium cyanide was chosen as the labeled starting material
(5) Tsai, A.-L.; Palmer, G.; Wu, G.; Peng, S.; Okeley, N. M.; van der
Donk, W. A.; Kulmacz, R. J. J. Biol. Chem. 2002, 277, 38311-38321.
(6) Brash, A. R. J. Biol. Chem. 1999, 274, 23679-23682;
(7) Kuhn, H.; Schewe, T.; Rapoport, S. M. AdV. Enzymol. Relat. Areas
Mol. Biol. 1986, 58, 273-311
(8) Sigal, E.; Grunberger, D.; Craik, C. S.; Caughey, G. H.; Nadel, J. A.
J. Biol. Chem. 1988, 263, 5328-5332.
(9) Brash, A. R.; Boeglin, W. E.; Chang, M. S. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 6148-6152.
(12) (a) Glickman, M. H.; Wiseman, J. S.;. Klinman, J. P J. Am. Chem.
Soc. 1994, 116, 793-794. (b) Hwang, C. C.; Grissom, C. B. J. Am. Chem.
Soc. 1994, 116, 795-796. (c) Glickman, M. H.; Klinman, J. P. Biochemistry
1995, 34, 14077-14092. (d) Glickman, M. H.; Klinman, J. P. Biochemistry
1996, 35, 12882-12892.
(13) Peng, S.; van der Donk, W. A. J. Am. Chem. Soc. 2003, 125, 8988-
8989.
(14) Lewis, E. R.; Johansen, E.; Holman, T. R. J. Am. Chem. Soc. 1999,
121, 1395-1396.
(15) Rickert, K. W.; Klinman, J. P. Biochemistry 1999, 38, 12218-12228.
(10) Young, R. N. Eur. J. Med. Chem. 1999, 34, 671-685.
(11) Cyrus, T.; Witztum, J. L.; Rader, D. J.; Tangirala, R.; Fazio, S.;
Linton, M. F.; Funk, C. D. J. Clin. InVest. 1999, 103, 1597-1604; Cyrus,
T.; Pratico, D.; Zhao, L.; Witztum, J. L.; Rader, D. J.; Rokach, J.; FitzGerald,
G. A.; Funk, C. D. Circulation 2001, 103, 2277-2282. Funk, C. D.; Cyrus,
T. Trends CardioVasc. Med. 2001, 11, 116-124. Cathcart, M. K.; Folcik,
V. A. Free Radical Biol. Med. 2000, 28, 1726-1734. Steinberg, D. J. Clin.
InVest. 1999, 103, 1487-1488. Belkner, J.; Stender, H.; Kuhn, H. J. Biol.
Chem. 1998, 273, 23225-23232.
350
Org. Lett., Vol. 6, No. 3, 2004

Scheme 4
Scheme 6
because of its commercial availability and relatively low cost.
Treatment of nitrile 3 with a saturated HCl/MeOH solution,
followed by hydrolysis yielded methyl ester 4 in good yield.
After deprotection and silylation, the methyl ester was
reduced to the aldehyde 2b using DIBAL-H.
Wittig olefination of aldehydes 2a and 2b with phospho-
nium salt 516 employing salt-free conditions17 resulted in
dienes 6 with 95:5 Z-selectivity (Scheme 4). The isomers
were not separated at this stage but carried on through the
synthesis; the minor isomers were removed by HPLC after
generation of the final arachidonic acid products. Deprotec-
tion of 6a and 6b using TBAF, followed by bromination
and refluxing with triphenylphosphine, afforded phospho-
nium salts 7a and 7b.
tyne with ethylmagnesium bromide and copper(I) bromide,
was reacted with bromide 10, yielding the skipped diyne 11
in 86% yield. Hydrogenation in the presence of nickel(II)
acetate yielded skipped diene 12, which was deprotected with
TBAF in acidic solution. The alcohol was subsequently
brominated and converted to phosphonium salt 7c.
The three isotopically labeled phosphonium salts 7a-c
were olefinated with aldehyde 134 to afford the labeled
methyl arachidonates 14 (Scheme 6). Hydrolysis with lithium
hydroxide followed by HPLC purification yielded the final
products, [10,10-2H2]- and [13,13-2H2]-arachidonic acid, and
[11-13C]-arachidonic acid.
The synthesis of [15-13C]-arachidonic acid was achieved
from commercially available [1-13C]-hexanoic acid. Follow-
ing esterification and lithium aluminum hydride reduction,
[1-13C]-hexanal was obtained by PCC oxidation. A Wittig
reaction between the labeled aldehyde 17 and triphenylphos-
phonium salt 16 yielded protected aldehyde 18 in modest
yield (Scheme 7). Deprotection with p-toluenesulfonic acid
Phosphonium salt 7c was required for the synthesis of
[13,13-2H2]-arachidonic acid. Its preparation started with the
protected alkyne 8 (Scheme 5). Deprotonation with ethyl-
Scheme 5
Scheme 7
magnesium bromide followed by addition to deuterium-
labeled paraformaldehyde produced the protected pent-2-yn-
1,5-diol 9 in good yield. This alcohol was reacted with carbon
tetrabromide and triphenylphosphine to yield propargyl
bromide 10. A copper acetylide, formed by treating 1-hep-
produced the â,γ-unsaturated aldehyde, which was reduced
to alcohol 19. Reaction with dibromotriphenylphosphorane
yielded labeled 1-bromo-non-3-ene, which was converted in
good yield to the phosphonium salt 20. We previously
(16) Pommier, A.; Pons, J.-M.; Kocienski, P. J. J. Org. Chem., 1995,
60, 7334-7339.
(17) (a) Greenwald, R.; Chaykovsky, M.; Corey, E. J. J. Am. Chem. Soc.
1963, 28, 1128-1129. (b) Bestmann, H. J.; Stransky, W.; Vostrowsky, O.
Chem. Ber. 1976, 109, 1694-1700.
Org. Lett., Vol. 6, No. 3, 2004
351

reported a facile and high yielding synthesis of skipped
aldehyde 21.4 Reaction of this aldehyde with phosphonium
salt 20 yielded [15-13C]-methyl arachidonate 7d. Final
hydrolysis with lithium hydroxide yielded the target com-
pound, [15-13C]-arachidonic acid 7d, in excellent yield.
Herein, we described the synthesis of four site-specifically
labeled arachidonic acids. The synthesis of both dideuterated
and 13C-labeled compounds was carried out in high efficiency
and isotopic purity. These compounds can be used as mech-
anistic probes for cyclooxygenase and lipoxygenases. Specif-
ically, the dideuterated compounds can be employed for ki-
netic isotope effect studies with 12- and 8-LOX, which both
abstract hydrogen atoms from C10, and for 15-LOX and
COX-1 and COX-2, which abstract a hydrogen atom from
C13. Mechanistic studies of the labeled substrates with these
enzymes are underway and will be published in due course.
Acknowledgment. This work was supported in part by
GM44911 from the National Institutes of Health. NMR
spectra were obtained in the Varian Oxford Instrument Center
for Excellence, funded in part by the W. M. Keck Founda-
tion, NIH (PHS 1 S10 RR10444), and NSF (CHE 96-10502).
Mass spectra were recorded on a Voyager mass spectrometer
funded in part by the National Institutes of Health (RR
11966).
Supporting Information Available: Experimental pro-
cedures and spectral characterization of all synthetic com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL0361711
352
Org. Lett., Vol. 6, No. 3, 2004