Structural characterization of a pentadienyl radical intermediate formed during catalysis by prostaglandin H synthase-2

Full text of DOI:10.1021/ja015599x

J. Am. Chem. Soc. 2001, 123, 3609-3610
3609
Scheme 1
Structural Characterization of a Pentadienyl Radical
Intermediate Formed during Catalysis by
Prostaglandin H Synthase-2
Sheng Peng,† Nicole M. Okeley,† Ah-Lim Tsai,‡ Gang Wu,‡
Richard J. Kulmacz,‡ and Wilfred A. van der Donk*,†
Department of Chemistry
UniVersity of Illinois at Urbana-Champaign
600 South Mathews AVenue, Urbana, Illinois 61801
DiVision of Hematology, Internal Medicine
UniVersity of Texas Medical School at Houston
6431 Fannin, Houston, Texas 77030
ReceiVed January 29, 2001
Scheme 2
Prostaglandin H synthase (PGHS) or cyclooxygenase (COX)
catalyzes the first committed step in the biosynthesis of all
prostaglandins and thromboxanes, the conversion of arachidonic
acid (1) into prostaglandin H2 (Scheme 1).1 These compounds
are important mediators in inflammation, and as such, the enzyme
has been of great interest for the development of antiinflammatory
agents.2 We report here the structural characterization of a radical
intermediate by electron paramagnetic resonance (EPR) spec-
troscopy in combination with stereospecifically deuterated sub-
strates.
The discovery of two isozymes in mammalian cells, COX1
and COX2,3 has allowed important improvements in nonsteroidal
antiinflammatory drugs (NSAIDs).4 COX1 is generally a consti-
tutively expressed protein, whereas COX2 expression is induced
in specific tissues in response to certain stimuli.5 Recently
developed COX2 selective inhibitors lack many of the toxic side
effects observed with nonselective inhibitors.6
The chemical mechanism for the conversion of arachidonic acid
to prostaglandin G2 in the cyclooxygenase reaction still holds
many unanswered questions. The overall mechanistic proposal
by Hamberg and Samuelsson in 19677 still largely stands, although
direct support for most of the steps and intermediates is lacking.
Important advances since 1967 have been the identification of a
tyrosyl radical as the initiator of catalysis,8,9 the detection of a
substrate-based radical by EPR spectroscopy (Scheme 2),9 and
very recently, the crystallographic characterization of substrate
and PGH2 bound to the enzyme.10 However, the precise structure
of the substrate radical needs further investigation. Tsai and co-
workers have proposed that the radical is a delocalized pentadienyl
radical spanning positions C11-C15 of arachidonic acid (i.e., I,
Scheme 2).9
Pentadienyl radicals have an odd-alternate spin distribution,
and therefore, the proposed radical in PGHS is expected to have
significant spin density at C11, C13, and C15. Site-specifically
deuterated arachidonic acids can contribute to corroboration of
the proposed structure, as their reaction with the enzyme should
lead to predictable changes in the hyperfine pattern observed in
the EPR spectrum of I. We therefore prepared (R)-[13-2H]-1, (R)-
[13,15-2H2]-1, and [15-2H]-1 as structural probes.
A chemoenzymatic synthesis of (R)-[13-3H]-1 has been reported
previously,11 but this route was not feasible for our purposes as
it would lead to partially deuterated products due to isotope
dilution during in vivo conversion of labeled stearate to arachi-
donic acid. Our entirely synthetic route was designed to allow
preparation of all three target compounds from one common
advanced intermediate, aldehyde 212 (Scheme 3). [15-2H]-1 was
prepared by Wittig reaction of 2 with phosphonium salt 3,
obtained in four steps from [1-2H]-hexanal. Arachidonic acid
stereospecifically deuterium-labeled at C13 was produced from
2 by conversion into phosphonium salt 4, followed by Wittig
olefination of aldehyde 5. This compound was prepared in three
steps from phosphonium salt 613 and hexanal. The doubly labeled
arachidonic acid, (R)-[13,15-2H2]-1, was prepared by the same
route using [1-2H]-hexanal. The isotopic purity of the synthetic
substrates was assessed by field ionization and electrospray mass
spectrometry,14 and the stereochemical purity of (R)-[13-2H]-1
† University of Illinois at Urbana-Champaign.
‡ University of Texas Medical School at Houston.
(1) Smith, W. L.; DeWitt, D. L.; Garavito, R. M. Annu. ReV. Biochem.
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(3) (a) Xie, W.; Chipman, J. G.; Robertson, D. L.; Erickson, R. L.;
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(4) (a) Vane, J. R.; Botting, R. M. Clinical Significance and Potential of
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L. J.; Kalgutkar, A. S. Curr. Opin. Chem. Biol. 1998, 2, 482-490.
(5) Herschman, H. R. Biochim. Biophys. Acta 1996, 1299, 125-140.
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M.; Rogers, R. S.; Rogier, D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.;
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Science 2000, 289, 1933-7. (b) Kiefer, J. R.; Pawlitz, J. L.; Moreland, K. T.;
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(14) Isotopic purity was determined by comparison of mass spectra obtained
for unlabeled and labeled synthetic material.
10.1021/ja015599x CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/23/2001

3610 J. Am. Chem. Soc., Vol. 123, No. 15, 2001
Communications to the Editor
Scheme 3
Figure 1. 9 GHz EPR spectra obtained by anaerobic incubation of COX2
(11-14 µM) with (A) unlabeled 1, (B) 15-[2H]-1, (C) 13(R)-[2H]-1, (D)
13,15-[2H2]-1, and (E) 5,6,8,9,11,12,14,15-[2H8]-1. All samples were
frozen approximately 60 s after addition of the substrate at 4 °C. The
overlaid spectra (- - -) show simulations. For instrument settings and
simulation parameters, see Supporting Information.
(R)-[13-2H]-1 provided a six-line signal (Figure 1C), whereas the
doubly labeled compound gave rise to a five-line signal (Figure
1D). These observations provide support for spin density at C13
and C15. Finally, reaction of octadeuterated arachidonic acid gave
rise to a five-line spectrum (Figure 1E), in good agreement with
the predicted changes for the proposed pentadienyl radical in
Scheme 2, as only two (at C11 and C15) of the eight deuteriums
are in positions that would alter the hyperfine pattern. It is
important to note that for all simulations in Figure 1 the
parameters, which are consistent with known pentadienyl radi-
cals,17 were kept invariant with the exception of adjusting the
size of the hyperfine value of protons substituted with deuter-
ons.9,16 Thus, the spectra in Figure 1 collectively provide strong
support for the assignment of the radical signal to a C11-C15
pentadienyl radical.18 This is direct19 experimental verification
of the structure of an intermediate in the original7 mechanism of
formation of prostaglandins by cyclooxygenase.
and the doubly labeled compound was determined by incubation
with soybean lipoxygenase. This enzyme converts 1 to 15-(S)-
hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-H-
PETE) by stereospecific abstraction of the pro-S hydrogen atom
from position 13 of the substrate.15 Comparison of the mass
spectra of the enzymatic products from labeled and unlabeled 1
gave an estimated lower limit of 96:4 and 98:2 er., respectively,
for the singly and doubly labeled arachidonic acids.
The three synthetically labeled substrates were used for
characterization of the radical signal observed previously by EPR
spectroscopy after anaerobic incubation of COX2 with arachidonic
acid.9 Similar to these earlier studies, unlabeled substrate resulted
in a seven-line signal (Figure 1A). Consistent with the proposal
that this signal derives from a pentadienyl radical spanning C11-
C15 (Scheme 2), substitution of the proton at C15 with a
deuterium resulted in a spectrum that can be simulated as a six-
line multiplet (Figure 1B).16 Similarly, incubation of COX2 with
Acknowledgment. Dedicated to the memory of Professor Jerry
Babcock. We thank Dr. Graham Palmer for use of his EPR instrument.
This work was supported by the National Institutes of Health (GM44911,
A.-L.T. and GM52170, R.J.K). N.M.O. thanks Abbott Laboratories for a
pre-doctoral fellowship.
(15) Hamberg, M.; Samuelsson, B. J. Biol. Chem. 1967, 242, 5329-35.
(16) This reduction in the multiplicity is the result of the substantially
2
1
smaller gyromagnetic ratio (γ) for H compared to H (γD/γH ) 0.15). The
triplet hyperfine splitting of the deuteron is too small to be resolved in these
spectra of frozen solutions, and hence, substitution of a proton with deuterium
results in the apparent loss of one hyperfine interaction.
(17) Davies, A. G.; Griller, D.; Ingold, K. U.; Lindsay, D. A.; Walton, J.
C. J. Chem. Soc., Perkin Trans. 2 1981, 633-641.
Supporting Information Available: Spectral data and experimental
procedures for all new compounds as well as a detailed description of
the determination of isotopic and optical purity of the final products (PDF).
This material is available free of charge via the Internet at http://pubs.acs.org.
(18) Exposure of the observed radical to O2 regenerates the tyrosyl radical,
consistent with its chemical competence in catalysis.9
(19) The same radical may have been detected indirectly in radical trap
studies: Schreiber, J.; Eling, T. E.; Mason, R. P. Arch. Biochem. Biophys.
1986, 249, 126-36.
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