Tyrosine-lipid peroxide adducts from radical termination: Para coupling and intramolecular Diels-Alder cyclization

Article abstract of DOI:10.1021/ja106503a

Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by

Full text of DOI:10.1021/ja106503a

Published on Web 11/19/2010
Tyrosine-Lipid Peroxide Adducts from Radical Termination:
Para Coupling and Intramolecular Diels-Alder Cyclization
Roman Shchepin,† Matias N. Mo¨ller,†,‡ Hye-young H. Kim,† Duane M. Hatch,†
Silvina Bartesaghi,§ Balaraman Kalyanaraman,| Rafael Radi,⊥ and Ned A. Porter*,†
Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt UniVersity,
NashVille, Tennessee 37235, United States, Facultad de Ciencias and Center for Free Radical
and Biomedical Research, UniVersidad de la Repu´blica, MonteVideo, Uruguay, Departamento de
Histolog´ıa and Center for Free Radical and Biomedical Research, Facultad de Medicina,
UniVersidad de la Repu´blica, MonteVideo, Uruguay, Department of Biophysics and Free Radical
Research Center, Medical College of Wisconsin, Milwaukee,
Wisconsin 53226, United States, and Departamento de Bioqu´ımica, Facultad de Medicina,
UniVersidad de la Repu´blica, MonteVideo, Uruguay
Received July 22, 2010; E-mail: n.porter@vanderbilt.edu
Abstract: Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine
gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The
novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass
spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable
peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts
have characteristic 13C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-
derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder
(IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl
13C chemical shifts at ∼198 ppm. All of the NMR HMBC and HSQC correlations support the structure
assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic
rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts
were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products
from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction
product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts
formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl
radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves
since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions
with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.
Introduction
for example, is thought to occur by a two-step reaction involving
the initial formation of a tyrosyl radical that subsequently reacts
Protein-derived tyrosine radicals can act as cofactors in some
enzymatic reactions, including those of class I ribonucleotide
reductase, prostaglandin H synthase, photosystem II,1,2 and
cytochrome C oxidase.3 On the other hand, protein-derived
tyrosyl radicals can also be formed in vivo as a result of
oxidative stress through mechanisms including both free radical4-7
and hemeperoxidase-dependent8 oxidations. Tyrosine nitration,
with nitrogen dioxide (Scheme 1).4,9-11 The formation of
3-nitrotyrosine is an important protein post-translational modi-
fication that can alter structure and function.12 It is associated
with acute and chronic disease states and can be a predictor of
disease risk and progression.11,13 The extent of protein tyrosine
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(5) Das, A. B.; Nagy, P.; Abbott, H. F.; Winterbourn, C. C.; Kettle, A. J.
Free Radical Biol. Med. 2010, 48, 1540.
† Vanderbilt University.
‡ Facultad de Ciencias and Center for Free Radical and Biomedical
Research, Universidad de la Repu´blica.
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§ Departamento de Histolog´ıa, and Center for Free Radical and Biomedi-
cal Research, Facultad de Medicina, Universidad de la Repu´blica.
| Medical College of Wisconsin.
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⊥ Departamento de Bioqu´ımica, Facultad de Medicina, Universidad de
la Repu´blica.
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Kalyanaraman, B.; Radi, R. Chem. Res. Toxicol. 2010, 23, 821.
(10) Bartesaghi, S.; Valez, V.; Trujillo, M.; Peluffo, G.; Romero, N.; Zhang,
H.; Kalyanaraman, B.; Radi, R. Biochemistry 2006, 45, 6813.
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10.1021/ja106503a  2010 American Chemical Society

Tyrosine-Lipid Peroxide Adducts from Radical Termination
A R T I C L E S
Scheme 1. Free-Radical-Mediated Tyrosine Oxidation Reactions
Scheme 2. Primary Coupling Products of Tyrosyl and Lipid Peroxyl
nitration depends on the particular protein structure and the
environment and location of the individual tyrosine residue.14
Tyrosyl radicals also couple to form 3,3′-dityrosine15-17 (see
Scheme 1), and the formation of tyrosine dimers is frequently
cited as evidence for the formation of tyrosyl radical species in
vivo. Dimerization has been associated with protein cross-linking
and the formation of high-molecular-weight species.16,17A recent
report, for example, showed that dimerization of a SLC30A
family of zinc transporters depends on redox-regulated covalent
tyrosine dimerization and suggested that dityrosine-dependent
membrane protein oligomerization may regulate the function
of diverse membrane proteins in normal and disease states.18
Most of the mechanistic studies of tyrosine nitration and
dimerization have been carried out under aqueous conditions,
but the same chemistry has been observed in tyrosine-containing
proteins that interact with membrane lipids or are located in
hydrophobic environments.14,19 It is important to note that
tyrosines are frequently observed in membrane-binding regions
of proteins, and a recent analysis has suggested that placement
of tyrosine residues in protein membrane locations minimizes
the free energy relative to their placement in solvent-exposed
or transitional sites.20 The colocalization of tyrosine protein
residues and membrane lipids suggests the possibility of
reactions of tyrosyl and lipid-derived radicals,9,10 since mem-
branes, particularly those containing polyunsaturated fatty acid
(PUFA)21 esters,22-24 are subject to peroxidation via chain-
carrying peroxyl free radicals. Membrane lipid peroxidation has
been linked to a number of human diseases, including
atherosclerosis25,26 and neurodegenerative disorders.27-30 It is
Radicals
notable that simultaneous lipid peroxidation, tyrosine dimer-
ization, and protein tyrosine nitration have been observed in
red blood cell membranes, indicating that these competing
processes occur even in membranes that contain R-tocopherol.31
Oxidation of tyrosine residues in ApoA1, a principal constitu-
ent of high-density lipoprotein (HDL), has been of particular
interest because HDL plays an important role in the transport
of cholesterol from peripheral tissues to the liver. Recent studies
have provided evidence that oxidative modification of ApoA1
leads to loss of HDL function.32,33 Modification of HDL
tyrosines by nitration or chlorination has been the principal focus
of most studies, but HDL tyrosines are also prime targets for
peroxidative modification since HDLs are lipid- and protein-
rich but poorly protected from peroxyl free radicals by nature’s
antioxidant, R-tocopherol.34,35
Here we report studies of lipid peroxyl coupling with
alkylphenoxy radicals that serve as models for tyrosyls (see
Scheme 2). The product mixture formed in a reaction of this
type is complex and includes dialkyl peroxide coupling com-
pounds as well as novel Diels-Alder adducts. It seems likely
that products similar to the ones identified here are major
products in the free radical peroxidation of tyrosine-containing
membranes, since cross-termination has been established as a
major pathway in radical reactions involving both peroxyl and
phenoxy species.36
(14) Bartesaghi, S.; Ferrer-Sueta, G.; Peluffo, G.; Valez, V.; Zhang, H.;
Kalyanaraman, B.; Radi, R. Amino Acids 2007, 32, 501.
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(18) Salazar, G.; Falcon-Perez, J. M.; Harrison, R.; Faundez, V. PLoS One
2009, 4, e5896.
Results
Co-oxidation of Hydrophobic Tyrosine (1) with 1-Palmitoyl-
(13S)-hydroperoxyoctadecadienoylglycerylphosphatidylcholine (2).
The hydrophobic tyrosine analogue10,37 N-t-BOC-L-tyrosine tert-
butyl ester (BTBE, 1; 2 mM) was mixed with enantiopure
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(21) Abbreviations: PUFA, polyunsaturated fatty acid; NMR, nuclear
magnetic resonance; MeOAMVN, 2,2′-azobis(4-methoxy-2,4-dimeth-
ylvaleronitrile); HPLC, high-performance liquid chromatography;
EtOAc, ethyl acetate; DMF, N,N-dimethylformamide; MS, mass
spectrometry; HSQC, heteronuclear single-quantum coherence; HMBC,
heteronuclear multiple-bond coherence; NOE, nuclear Overhauser
effect; NOESY, NOE spectroscopy; ESI, electrospray ionization; TIC,
total ion current; m/z, mass-to-charge ratio; IMDA, intramolecular
Diels-Alder; BTBE, N-t-BOC-L-tyrosine tert-butyl ester; PLPC,
1-palmitoyl-2-linoleoylglycerylphosphatidylcholine.
(29) Giasson, B. I.; Duda, J. E.; Murray, I. V. J.; Chen, Q.; Souza, J. M.;
Hurtig, H. I.; Ischiropoulos, H.; Trojanowski, J. Q.; Lee, V. M. Science
2000, 290, 985.
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A R T I C L E S
Shchepin et al.
Figure 1. HPLC-MS analysis of the reaction mixture for the reaction of BTBE (1) and PLPC-(13S)-OOH (2). (A) Total ion current of the reaction mixture.
The early-eluting peak at 8.2 min is 1, and the later-eluting peak at 18.0 min is 2. Reversed-phase HPLC on C-18 with solvents A (10 mM NH4OAc in H2O)
and B (10 mM NH4OAc in 95% CH3OH/H2O) was used, with the gradient starting at 70% B, going to 100% B over 10 min, and remaining at 100% B for
15 min. (B) Extracted ion current detecting the adduct 3 (m/z 1125.5).
PLPC-(13S)-hydroperoxide (2; 6.8 mM) generated by reaction
of the 2-linoleoylglycerophospholipid with soybean lipoxyge-
nase. The mixture was sonicated at room temperature to form
micelles in the presence of the azo free radical initiator 2,2′-
azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeOAMVN; 1
mM). The micellar solution was allowed to stand in a 37 °C
sand bath for 4 h and then analyzed by HPLC-MS. A typical
chromatogram is presented in Figure 1.
led to a series of reactions of the azo-initiated co-oxidation of
phenol 4 with the symmetrical diene (6E,9E)-pentadecadiene
(5).38
MS analysis indicated the formation of adduct(s) having a
parent ion at m/z 1125.5, corresponding to the expected mass
of adduct 3. However, attempts to isolate and further characterize
the adduct(s) proved futile because the initial products were
only moderately stable and appeared to undergo further trans-
formations upon attempted purification. Furthermore, the yield
of the m/z 1125 adduct(s) formed under the micellar conditions
was low, and the products were not well-behaved on silica gel.
For these reasons, we sought to simplify both the tyrosine
(phenol) and membrane lipid reactants to allow any lipid-
tyrosine adducts formed to be isolated on a scale that would
permit characterization of the products by the normal tools of
organic chemistry, including two-dimensional (2D) NMR
spectroscopy.
Peroxide Adducts of Peroxyl-Phenoxy Coupling. UV-active
tyrosine analogue 4 was co-oxidized with diene 5 in benzene
for 9 h at 37 °C using MeOAMVN as the initiator. The reaction
progress was monitored at 330 nm by normal-phase (NP)
HPLC-UV using 25% ethyl acetate in hexanes as the mobile
phase (Figure 2A). In the early stages of oxidation, a peak at
16 min appeared (fraction IV), while additional peaks were
detected at longer reaction times. The peak at 16 min (Figure
2A, fraction IV) was collected, evaporated to dryness, and
resuspended in 1:1 H2O/acetonitrile. Reversed-phase (RP) HPLC
of fraction IV (Figure 2B) showed it to be homogeneous; the
1
purified compound was dissolved in CDCl3, and H, COSY,
NOESY, HSQC, HMBC, and 13C spectra were obtained at room
temperature. The spectral data acquired for fraction IV led to
the assignment of structure 6 for this major product of the co-
oxidation reaction. Compound 6 has a characteristic 13C
chemical shift for the carbonyl carbon (C4) at 185 ppm, and in
the HMBC experiment, C4 exhibited only one three-bond cross-
peak to H2. Extraction of coupling constants suggested that the
conjugated diene has E,E geometry, and the integration and
coupling pattern were consistent with the proposed peroxyl-
phenoxy para-coupling product.
Examination of the reactions of several tyrosine derivatives
with the simplest oxidizable fatty acids (or esters) still gave
complex mixtures of products that were difficult to purify. The
final simplification of the tyrosine model was to reduce the
complexity by using the phenol (7-methoxy-2-oxo-2H-chromen-
4-yl)methyl 3-(4-hydroxyphenyl)propanoate (4), a compound
that has no stereogenic center but does possess a built-in
chromophore unrelated to the oxidizable tyrosine moiety. The
compound, which was prepared in one step in 87% yield,
provided a model substrate having λmax ) 330 nm that could
be used as a tag for products derived from the phenol. The fatty
acid component was also simplified in order to minimize the
number of isomeric products generated during the oxidation
reaction. This reduction of complexity of the system studied
The adduct 6 was found to be unstable, and it formed a
mixture of stereoisomeric products with structures assigned as
the intramolecular Diels-Alder (IMDA) cyclization products
7a-c. The same NP-RP protocol used to purify 6 was also
used to isolate the stereoisomeric IMDA products. Thus, the
(37) Zhang, H.; Joseph, J.; Feix, J.; Hogg, N.; Kalyanaraman, B. Biochem-
istry 2001, 40, 7675.
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Tyrosine-Lipid Peroxide Adducts from Radical Termination
A R T I C L E S
showed it to be a mixture of compounds that are isomeric with 6
at the conjugated diene center adjacent to C6′. The suggested
structures have one of the double bonds of the conjugated structure
in the Z configuration. The MS and UV analyses of fraction III
are consistent with the structures of the proposed constituents.
Distinctive differences between the para-coupling and IMDA
adducts can be seen not only in the 13C chemical shifts but also
in the HMBC correlations. As noted above, the 13C chemical
shift for the carbonyl carbon (C4) of 6 was observed at 185
ppm, while those of the IMDA products were generally seen at
199 ppm. In the HMBC experiment, C4 of 6 exhibited only
one three-bond cross-peak to H2, whereas each cyclic adduct
gave rise to two additional cross-peaks to the ring-junction
protons, as shown in Figure 3. The stereochemistry of each
IMDA adduct was assigned on the basis of NOESY correlations
between protons that are within the allowed spatial vicinity.
HSQC spectra provide one-bond correlations between carbons
and protons along with the number of attached protons. The
13C signals of the peroxide-bearing carbons of 6 and the IMDA
adducts showed only one HSQC cross-peak, corresponding to
C6′ at 86.0 ppm for 6 and 82.2 ppm for the diastereomeric
adducts 7. Any ortho-coupling adducts such as 8 or potential
derivative IMDA products such as 9 and 1039-43 would exhibit
two HSQC cross-peaks for both C5 and C6′ in each adduct.
We therefore conclude that significant amounts of stable ortho-
coupling products were not formed. Both the HSQC and HMBC
spectra therefore support the notion that para coupling of the
phenoxy and peroxyl radicals occurs, forming adducts 6 and
7a-c as the major stable adducts. A more detailed discussion
of the NMR analyses is presented in the Supporting Information.
Figure 2. HPLC-UV (330 nm) analysis of the co-oxidation reaction of 4
and 5. (A) Normal-phase separation of the reaction mixture into fractions
I-IV (4 elutes at 23 min). (B-D) Reversed-phase analyses of the separated
fractions IV, II, and I.
reaction mixture was first purified with NP HPLC to collect
the acyclic and cyclic adduct fractions, and the peaks collected
as fractions I-IV were then analyzed for purity with RP HPLC.
Fraction I contained only one product, 7a, which eluted at 14.3
min in the RP HPLC step (Figure 2D), and fraction II separated
into two peaks that eluted at 14.1 (7b) and 14.9 (7c) min. (Figure
2C). It is noteworthy that all of the IMDA products 7a-c were
formed from the single peroxide precursor 6 (i.e., fraction IV).
Silver-Coordinated (LC-Ag+)-CID-MS Analysis of
Acyclic and Cyclic Adducts. While molecular-ion MS informa-
tion for the adducts was collected using electrospray ionization
(ESI) and atmospheric pressure chemical ionization (APCI)
methods, good collision-induced dissociation (CID) spectra
could not be obtained by these conventional approaches. Silver
ion forms stable complexes with unsaturated compounds, and
silver-ion-coordination MS proved to be a useful approach to
provide additional support for the assigned structures of 6 and
7. For all of the isolated adducts, precursor masses of at m/z
699 and 701 [M + Ag] due to the two isotopes 107Ag and 109Ag
were observed (Figure 4A). The silver ion complex of the
primary adduct 6 displayed a major fragment ion at m/z 476
[M - 223 + Ag] due to cleavage of the peroxide bond (Figure
(39) Masuda, T.; Bando, H.; Maekawa, T.; Takeda, Y.; Yamaguchi, H.
Tetrahedron Lett. 2000, 41, 2157.
(40) Masuda, T.; Maekawa, T.; Hidaka, K.; Bando, H.; Takeda, Y.;
Yamaguchi, H. J. Agric. Food Chem. 2001, 49, 2539.
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Fraction III could not be separated completely from fraction IV
and appeared to be a product closely related to 6. Thus, NMR
analysis of fraction III (Figure S5 in the Supporting Information)
(43) Masuda, T.; Yamada, K.; Maekawa, T.; Takeda, Y.; Yamaguchi, H.
J. Agric. Food Chem. 2006, 54, 6069.
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Shchepin et al.
Figure 3. Representative HMBC correlations in adducts 6 and 7b. (left) Adduct 6: C4 (185 ppm) to H2 (6.8 ppm). (right) Adduct 7b: C4 (199 ppm) to H2
(6.8 ppm) and H6 (2.9 and 2.8 ppm). There is also a weak two-bond correlation to H5 with this diastereomer.
Figure 4. Representative MS spectra of primary and IMDA adducts in 1 µM AgNO3 in MeOH sprayed directly into the MS source (collision pressure, 1.40
mTorr; collision dissociation energy, 25 eV): (A) precursor masses of the primary and IMDA adducts [M+Ag] at m/z 699 and 701; (B) CID of the silver
ion complex of primary adduct 6 at m/z 699; (C) CID of IMDA adduct 7b.
4B). On the other hand, the silver ion complex of the cyclic
adduct exhibited multiple fragmentations, as assigned in Figure
4C.
Kinetics of the Intramolecular Diels-Alder Reaction of
Adduct 6. Rate constants for the IMDA reaction under several
sets of conditions were determined. The transformation was
monitored at 231 nm, the λmax of the conjugated diene (7′E,9′E)-
pentadecadiene. The decrease in absorbance at 231 nm was due
to loss of the conjugated diene as the reaction proceeded, as
shown in Figure 5. The data fit a single-exponential decay,
We found no evidence for the formation of o-hydroquinone 12
in the crude product mixture formed from co-oxidation of 4
and 5, which also suggests that coupling of the peroxyl radical
indicating a first-order process with a rate constant (k) of (9 (
is exclusively para. Cuprous ion was found to be the most
1) × 10-5 s-1 in ethanol at 37 °C. The rate constant was lower
effective way to reduce 6, while ferrous salts, thiourea, and
in aprotic solvents such as acetonitrile [k ) (6.8 ( 0.8) × 10-5
glutathione generated some compound 11, albeit in low yield.
s-1] and hexanes [k ) (1.3 ( 0.3) × 10-5 s-1] at 37 °C, also
The p-OH product 11 was independently synthesized by the
having an apparent correlation with polarity. Eyring analysis
reaction of phenol precursor 4 with PhI(OAc)2.44,45 The isomeric
hydroquinone 12 was also independently synthesized, so
authentic standards of both products were available for com-
parison.
gave ∆Hq ) 68 kJ mol-1 and ∆Sq ) -98 J K-1 mol-1 in ethanol
in the 25-50 °C range.
Reduction of Adduct 6. While a rich chemistry is expected
for 6, we limited the scope of our current studies to an
exploration of reduction reactions leading to stable end products.
Thus, reaction of 6 with cuprous ion or thiourea gave the
p-hydroxycyclohexadienone 11 in modest yield:
(44) Wipf, P.; Kim, Y. Tetrahedron Lett. 1992, 33, 5477.
(45) Pelter, A.; Satchwell, P.; Ward, R. S.; Blake, K. J. Chem. Soc., Perkin
Trans. 1 1995, 2201.
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Tyrosine-Lipid Peroxide Adducts from Radical Termination
A R T I C L E S
Figure 5. Kinetics of the intramolecular Diels-Alder reaction of adduct 6 in ethanol at 37 °C. (A) Spectra collected every 1 h. The inset shows the
difference between the spectra recorded at t ) 7 h and the initial time. (B) Fit of the decay at 231 nm to a single-exponential function, indicating a first-order
reaction with rate constant k ) (9 ( 1) × 10-5 s-1 (n ) 3). The absorbance at 320 nm (coumarin moiety) remained constant and was used as a normalizing
element.
Co-oxidation of Tyrosine Derivatives with Methyl
Linoleate. The formation and isolation of phenoxy-peroxyl
radical cross-termination products in the simple model system
served as a template for the reaction of more complex systems,
including N-acetyltyrosine ethyl ester 13 and the tyrosine-alanine
dipeptide 14. Reaction of both substrates with methyl linoleate
gave rise to adducts that had the mass spectra appropriate for
the tyrosyl-linoleate peroxyl radical cross-termination product,
and the dipeptide system was subjected to a more detailed
analysis. The primary product of co-oxidation of methyl linoleate
and dipeptide 14 had a UV λmax at 235 nm, indicating the
presence of a conjugated diene; the molecular ion was observed
at m/z 633.4 (Figure S13 in the Supporting Information), and
NMR analysis indicated the presence of a major product or
mixture of major products having a 13C chemical shift for a
carbonyl carbon at ∼185 ppm and 2D spectra consistent with
the formation of a product or products having a structure
analogous to 6 (Figure S15 in the Supporting Information). It
should be noted that four linoleate hydroperoxide stereoisomers
are formed in the peroxidation reaction, and four coupling
products with structures analogous to 6 are thus likely to be
formed. This accounts for the complexity of the product mixture
as analyzed by NMR spectroscopy and HPLC. Products were
also found in the reaction mixture of 14 and methyl linoleate
that had a 13C chemical shift for a carbonyl carbon at 196-198
ppm and 2D spectra consistent with formation of a product or
products having structures analogous to 7a-c.
for the [M + 18] adduct (D) before and (E) after reaction with
CuCl. Chromatogram B shows a synthetic standard of o-
hydroquinone 16, and the synthetic p-hydroxytyrosyl dipeptide
15 is shown in chromatogram C. Clearly, there is no evidence
for the formation of the hydroquinone that would be produced
by ortho coupling. In summary, all of the data gathered from
Reaction of the linoleate-peroxyl adduct mixture of 14 with
CuCl gave rise to the 4-hydroxycyclohexa-2,5-dione 15 (Figure
S18 in the Supporting Information), a product that was
synthesized independently by reaction of 14 with PhI(OAc)2.44,45
The chromatogram shown in Figure 6 illustrates the fact that
the para-coupling product is the only adduct formed. Chro-
matograms D and E in Figure 6 show an analysis of the reaction
product formed from dipeptide 14 and methyl linoleate scanned
Figure 6. Extracted ion chromatograms of the CuCl reduction mixture of
14-linoleate adducts: (A) 14; (B) synthetic 16; (C) synthetic 15; (D) crude
co-oxidation mixture before reduction; (E) crude co-oxidation mixture after
CuCl reduction.
9
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A R T I C L E S
Shchepin et al.
Scheme 3. Radical Termination Products of p-Cresol and tert-Butyl Hydroperoxide
the reaction of 14 and methyl linoleate are consistent with the
formation of para cross-termination primary products in this co-
oxidation.
the knowledge gained from this study could be used in studies
of relevant biological systems.
The free-radical-initiated reaction of the hydrophobic tyrosine
BTBE and the phospholipid hydroperoxide PLPC-(13S)-OOH
did indeed give a product fraction having the correct m/z in the
precursor-ion mass spectrum as well as an MS2 fragmentation
pattern consistent with an adduct of tyrosine and peroxide. The
isolation of homogeneous products from BTBE proved difficult,
but co-oxidation of simple phenol 4 and symmetrical diene 5
did give sufficient quantities of adducts for a comprehensive
NMR study. The major product identified at early stages of the
reaction was the peroxide para-coupling product 6, with no
evidence for the formation of the phenol dimer or ortho-coupling
products. The peroxide 6 slowly undergoes an intramolecular
Diels-Alder reaction to give at least three tricyclic products,
with one of the endo stereoisomers, 7b, as the major IMDA
product formed. We note that while the half-life for IMDA
cyclization at 37 °C is on the order of 2 h, the rate is solvent-
dependent and likely subject to acid catalysis. Of particular note
is the fact that the IMDA reaction leads to new C-C bonds
between the lipid and phenol (or tyrosine). Thus, while the
peroxide bond of a lipid-protein adduct such as 6 should be
readily reduced and the lipid-protein bond severed, any IMDA
lipid-protein coupling products formed are more likely to be
of a permanent nature.
Discussion
Phenolic antioxidants trap peroxyl radicals in a two-step
sequence: the first step is an H-atom transfer from phenol to
the peroxyl radical (eq 1),
kinh
ArOH + ROO• 8 ArO• + ROOH
(1)
and the second is the reaction of the intermediate phenoxy
radical with another peroxyl radical to give nonradical products
(eq 2).
ArO• + ROO• f nonradical products
(2)
Nature’s phenolic antioxidant, R-tocopherol, reacts with peroxyl
radicals with a rate constant for inhibition (kinh) of over 2 ×
106 M-1 s-1, and the second step, a radical-radical coupling,
occurs with a rate close or equal to the diffusion-controlled
value. The inhibition rate constant kinh depends dramatically on
the nature of the substituents on the phenol. Tyrosine, a para-
substituted alkylphenol, has a bond dissociation enthalpy of 85.2
kcal/mol,46 which is nearly 9 kcal/mol higher than that of
tocopherol, and as expected, the inhibition rate constant recently
reportedfortyrosineissubstantiallylessthanthatoftocopherol.9,47
Nevertheless, as a p-alkylphenol, tyrosine is expected to be a
modestly active antioxidant and trap peroxyl radicals to give
nonradical termination products.
The phenolic radical termination step leading to nonradical
products has been studied in some detail for R-tocopherol as
well as for commercial products such as butylated hydroxy-
toluene (BHT), butylated hydroxyanisole (BHA), and simple
phenols.36 The termination products observed are usually the
result of peroxyl coupling at the para and ortho positions of the
aryloxy ring. Simple phenols such as p-cresol give cross-
coupling products with tert-butylperoxyl radicals bonded ex-
clusively at the para position (see Scheme 3). Products from
aryloxy radical dimerization are also formed, but the cross-
coupling product 17 is the major product formed under most
reaction conditions.36 In view of this precedent, it is notable
that cross-termination products of tyrosine, itself a p-alkylphenol,
and lipid peroxyl radicals have not been previously detected in
extracts from cells, tissues, and biological fluids. One suspects
that the primary peroxide coupling products may not survive
biological reducing agents present in fluids and tissues, and for
this reason, we chose to examine tyrosyl-peroxyl radical
coupling at the fundamental chemical level with the idea that
Peroxyl radical coupling with phenoxy radicals followed by
IMDA cycloaddition has been reported in the reactions of
curcumin39,40 as well as caffeic41 and ferulic acid42,43 esters.
Caffeic acid, curcumin, and ferulic acid have phenol ring
substitution as in 18, and the products that have been isolated
appear to be the result of ortho coupling to the phenolic OH.
The preference for ortho coupling in 18 is likely due to the fact
that para coupling in 18 would disrupt the olefin conjugation;
this is not the case for tyrosine, which has para alkyl substitution.
The system studied in detail, 4 + 5 f 6, does serve as a
model for the reactions of systems more relevant to proteins
and fatty acids. Thus, the data accumulated from the reactions
of 13 or 14 with methyl linoleate gave evidence of a
linoleate-tyrosine para adduct. While the product mixture was
complex and adducts could not be isolated in pure form,
characteristic MS, MS2, and NMR data were acquired and
indicated that the chemistry observed in the model system
translates to the more complex peptide-lipid reactions. Evidence
for the formation of para adducts and reduction of the primary
products to the 4-hydroxycyclohexa-2,5-dienone was obtained.
(46) Nara, S. J.; Valgimigli, L.; Pedulli, G. F.; Pratt, D. A. J. Am. Chem.
Soc. 2010, 132, 863.
(47) Jha, M.; Pratt, D. A. Chem. Commun. 2008, 10, 1252.
9
17496 J. AM. CHEM. SOC. VOL. 132, NO. 49, 2010

Tyrosine-Lipid Peroxide Adducts from Radical Termination
Scheme 4. Tyrosine Lipid-Mediated Protein Cross-Link
A R T I C L E S
We note that both the lipid peroxide-tyrosine adducts, any
derived 4-hydroxycyclohexadienones, and the IMDA cycload-
ducts are electrophiles and should be subject to attack by
nucleophiles from proteins or nucleic acids. Cross-conjugated
cyclohexadienones are prone to undergo Michael addition, and
cysteines, lysines, and histidines are potential protein-derived
nucleophiles. A Michael addition of a cysteine on one protein
and a tyrosine-derived electrophile on another would lead to a
protein-lipid-protein adduct, which could account for the
formation of high-molecular-weight protein species in systems
under conditions of oxidative stress, as shown in Scheme 4.
High-molecular-weight protein-protein adducts are fre-
quently found under conditions of oxidative stress, and
tyrosyl-tyrosyl radical coupling has been cited as one mech-
anism for formation of these adducts.8,18,19,25,32,33,48-51 We
suggest that lipid peroxide-tyrosine adducts such as those
reported here are also likely sources of such high-molecular-
weight protein species under conditions of oxidative stress.
Oxidizable lipids are likely to be present in great excess relative
to tyrosine residues in biological membranes, which are known
targets of reactive oxygen species. The formation of lipid-
derived peroxyl radicals is in fact one of the cornerstone
reactions of oxidative stress, and these lipid peroxyls are
relatively mobile in a membrane in comparison with a tyrosine
protein residue.52
Tyrosine radical-peroxyl radical chemistry is likely to be
dependent on the local protein-lipid environment, including
the presence or absence of antioxidant inhibitors. Tyrosine
gives up its phenolic hydrogen some 3 orders of magnitude
slower than does R-tocopherol, so it seems likely that tyrosine
radical chemistry will be suppressed if tocopherol is present
in a lipid membrane or compartment. However, circumstances
exist where tocopherol intervention in tyrosine-lipid free
radical chemistry seems unlikely, one such instance being
in HDL biology. HDL plays an important role in reverse-
cholesterol transport, and oxidative modification of HDL
particles has been suggested to play a role in the development
of cardiovascular disease.19,32,34,48,49,51,53,54 An average HDL
particle has a molecular weight of 270 kD, of which 50% is
protein and 28% phospholipid. HDL contains on average
∼0.67 molecules of R-tocopherol per particle.53 This means
that one in three HDL particles should have no R-tocopherol,
while each should contain ∼98 molecules of phospholipid
and 30-40 tyrosine residues (see the Supporting Information
for calculations that lead to this estimate).34,53 In those
particles that contain no R-tocopherol, unsaturated lipids and
tyrosine protein residues are among the most likely targets
of peroxyl radical attack.
Once membrane or lipoprotein tyrosyl radicals are formed,
their fate is determined by whether the radical first encounters
another tyrosyl radical and undergoes self-termination to give
a tyrosine-tyrosine dimer or instead finds a lipid peroxyl
radical to give a cross-termination product. Our studies in
homogeneous solution found significant amounts of cross-
termination products under conditions where the oxidizable
lipid is present in excess relative to the phenol. It seems
reasonable to suggest that many biological membranes and
lipid particles provide local environments in which tyrosyl-
tyrosyl radical coupling is limited by the specific sites and
rate of diffusion of these protein radicals. Lipid peroxyl
radicals, on the other hand, would likely be substantially more
mobile than protein tyrosine radicals, perhaps by 2 or 3 orders
of magnitude, and this should promote the cross-termination
reaction.52,55-58
Finally, we note that the focus of a host of studies on protein
tyrosine modification has mainly centered on ortho substitution
(i.e., chlorination, hydroxylation, and nitration). The stable lipid
peroxide-tyrosine adducts identified here resulted exclusively
from para addition to the tyrosine moiety, which suggests that
para-substituted products may also be formed in other radical
transformations. Such para-addition compounds would be active
electrophiles and subject to reaction with protein and nucleic
acid nucleophiles. Numerous reports have suggested that oxida-
tion and adduction of tyrosines might play an important role in
numerous diseases, including atherosclerosis25,26,33,59,60 and
neurodegenerative disorders such as Parkinson’s and Alzhe-
imer’s diseases.27-31,61-75 We suggest that lipid and lipid
peroxyl radicals76 may play an important role in this chemistry.
Experimental Section
General Methods and Materials. BTBE was prepared by Dr.
Joy Joseph of the Medical College of Wisconsin. The initiator
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9
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A R T I C L E S
Shchepin et al.
1
MeOAMVN was purchased from Wako Chemicals USA, Inc.
(Richmond, VA) and dried under high vacuum for 2 h and kept at
-80 °C until use. Methyl linoleate and linoleic acids were purchased
from NuChek Prep (Elysian, MN). Symmetric (6E,9E)-pentadeca-
diene was synthesized following the method reported previously.38
Benzene (HPLC grade) was passed through a column of neutral
alumina and stored over molecular sieves. Hexanes, ethyl acetate,
methylene chloride, tetrahydrofuran (THF), and methanol (all
99.9%) were purchased from Thermo Fisher Scientific Inc. Lipids
were obtained from Avanti Polar Lipids, and 3-(4-hydroxyphenyl)-
propionic acid and 4-bromomethyl-7-methoxycoumarin were pur-
chased from TCI America. 4-Dihydroxyhydrocinnamic and L-ala-
nine methyl ester hydrochloride were purchased from Sigma-
Aldrich. N-Acetyl-L-tyrosine was purchased from Fluka. PhI(OAc)2
(iodobenzene diacetate) was purchased from Acros Organics.
Triethylamine and N,N-dimethylformamide (DriSolv) were pur-
chased from EMD and used without further purification. All
glassware was oven-dried prior to use.
HPLC Analysis Conditions. NP HPLC analyses were performed
using a Beckman Ultrasphere 5 µm silica column (4.6 mm × 25
cm) using different methods: condition A: isocratic method using
20% ethyl acetate in hexanes at a flow rate of 1.5 mL/min; condition
B: 10% isopropyl alcohol in hexanes increasing to 15% isopropyl
alcohol over 15 min and remaining there for 5 min; condition C:
8% isopropyl alcohol in hexanes at 4 mL/min using a semiprepara-
tive NP column (Beckman Ultrasphere Silica 5 µm, 10 mm × 25
cm). RP HPLC analyses were performed using a Luna C-18 column
(4.6 mm × 25 cm) from Phenomenex (Torrance, CA). The mobile
phases used in RP HPLC were: condition 1: (A) 10 mM ammonium
formate and (B) acetonitrile, with an initial gradient of 80% B that
was increased to 95% B immediately followed by 99% B for 10
min; condition 2: (A) 10 mM NH4OAc in H2O and (B) 10 mM
NH4OAc in 95% CH3OH/H2O, with a gradient that started with
70% B, was increased to 100% B over 10 min, and was maintained
at 100% B for 15 min; condition 3: (A) 10 mM ammonium formate
and (B) acetonitrile, with a gradient that started at 25% B for 5
min, was increased to 50% B for 5 min and then to 75% B for 5
min, which was followed immediately by 99% B for 5 min;
condition 4: starting with 70% acetonitrile/10 mM ammonium
formate, increasing to 83% acetonitrile over 20 min, and then
returning to 70% acetonitrile.
MHz. H spectra were acquired in 2.5 mm NMR tubes using a
Bruker 5 mm cryogenically cooled NMR probe. Chemical shifts
were referenced internally to chloroform (7.26 ppm), which also
served as the 2H lock solvent. 1H, 2D COSY, NOESY, HSQC, and
HMBC spectra were obtained for all of the adducts for structural
assignments. 13C spectra were obtained only for the major adducts.
Some NMR spectra were interpreted using MestReNova NMR
6.1.1.
Silver-Coordinated (LC-Ag+)-CID-MS of Acyclic and
Cyclic Adducts. The adducts were purified by HPLC prior to MS
analysis and then dissolved in 0.3 mM AgNO3 solution in MeOH
to obtain MS2 information.77 MS was performed using a Thermo
Finnigan TSQ Quantum Ultra instrument equipped with a Finnigan
Surveyor Autosampler Plus (San Jose, CA). Nitrogen gas served
both as the sheath and auxiliary gas, and argon served as the
collision gas. The electrospray needle was maintained at 4.6 kV,
and the heated capillary temperature was 200 °C. CID of the
tyrosine adducts was set at either 25 or 35 eV under 1.4 mTorr of
argon. In the displayed spectra, the scans were averaged across the
individual chromatography peaks. Data acquisition and analysis was
performed using Xcalibur software, version 2.0 SR2 (San Jose, CA).
HPLC-ESI Mass Spectrometry. HPLC-MS/MS analysis was
performed on a Waters Aquity UPLC system (Waters, Milford, MA)
connected to a Finnigan LTQ mass spectrometer (Thermo Fisher
Scientific, Waltham, MA) operating in the ESI positive-ion mode.
The same HPLC mobile phases and gradients described above were
used for the LC-MS analysis, but a different column, Luna C8
with 3 µm at a flow rate of 350 µL/min, was employed. MS analysis
was performed in both data-dependent and selected-ion-monitoring
(SIM) mode for the desired m/z.
Synthesis of PLPC-(13S)-OOH (2). PLPC (5 mg) and 4 mg of
soybean lipoxygenase (Sigma, St. Louis, MO) were dissolved in 5
mL of 100 mM borate/10 mM deoxycholic acid buffer (pH 9). The
reaction mixture was stirred vigorously at room temperature for
1 h, after which 2:1 CHCl3/CH3OH was used twice (3 mL each) to
extract PLPC and PLPC-OOH. The organic layer was collected
and the solvent evaporated using a rotary evaporator. The phos-
pholipid extracted in this way was resuspended in CH3OH and
purified with HPLC using 95% CH3OH in H2O as the solvent. The
collected peak was dried and resuspended in isopropyl alcohol for
use as a stock solution (2.16 mg/4 mL).
Oxidation of BTBE (1) and PLPC-(13S)-OOH (2) in a
Liposome. BTBE (33.7 mg) was dissolved in 1 mL of CH3OH to
prepare a 100 mM stock solution. PLPC-(13S)-OOH stock solution
(1 mL, 540 µg of 2) and BTBE stock solution (2 µL) were combined
in an Eppendorf tube, and 2 µL of MeOAMVN stock solution (50
mM in benzene) was added. The solution was vortexed for 2 min
and then dried under a nitrogen stream. NH4OAc (100 µL of a 100
mM solution, pH 7) was added to the dried mixture to make a
solution with final 2, 1, and MeOAMVN concentrations of 6.8, 2,
and 1 mM respectively. The reaction mixture was sonicated for 5
min and then allowed to stand in a 37 °C sand bath with stirring.
An aliquot (30 µL) was taken from the reaction mixture after 4 h
and diluted with 100 µL of CH3OH for MS analysis.
Synthesis of Tyrosine Analogue 4. To a 100 mL round-bottom
flask was added 3-(4-hydroxyphenyl)propionic acid (0.62 g, 3.72
mmol), and the flask was purged with argon. DMF (40 mL) and
then 4-bromomethyl-7-methoxycoumarin (1.0 g, 3.7 mmol) fol-
lowed by triethylamine (0.52 mL, 3.7 mmol) were added. The flask
was wrapped with aluminum foil and stirred at room temperature
under an argon balloon for 24 h. The solvent was then removed in
vacuo, yielding the crude product as a pale-brown oil. Silica flash
column chromatography (60:39:1 hexanes/EtOAc/TEA) afforded
the product, 4, as a white solid (1.15 g, 87%) upon solvent removal.
1H NMR (400 MHz, DMSO): δ 9.17 (s, 1H), 7.58 (d, J ) 8.9 Hz,
1H), 7.05-6.98 (m, 3H), 6.94 (dd, J ) 8.9, 2.5 Hz, 1H), 6.72-6.59
NMR Studies. All of the reported NMR spectra were acquired
using a 14.1 T Bruker magnet equipped with a Bruker DRX
spectrometer operating at a proton Larmor frequency of 600.13
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Tyrosine-Lipid Peroxide Adducts from Radical Termination
A R T I C L E S
(m, 2H), 6.23 (s, 1H), 5.31 (d, J ) 1.1 Hz, 2H), 3.85 (s, 3H), 2.75
(tt, J ) 6.8, 3.4 Hz, 4H). 13C NMR (101 MHz, DMSO): δ 172.29,
162.90, 160.29, 156.03, 155.35, 150.72, 130.69, 129.49, 126.20,
115.48, 112.69, 110.67, 109.58, 101.38, 61.54, 56.35, 35.60, 29.78.
HRMS (M + Na) m/z: calcd, 377.0996; found, 377.0996.
Oxidation of Tyrosine Analogue 4 with (6E,9E)-Penta-
decadiene (5). To a solution of 5 (0.5 g, 2.4 mmol) in benzene (3
mL) at 37 °C were added a solution of 4 (21 mg, 0.06 mmol in 2
mL of MeCN) and the radical initiator MeOAMVN (30 mg, 0.1
mmol). The reaction mixture was kept at 37 °C under an oxygen
atmosphere for 9 h. Reaction progress was monitored by HPLC
after 5 h of oxidation. The reaction was stopped at 9 h to obtain
pure acyclic adduct 6 for NMR experiments (Table S1 in the
Supporting Information). HRMS (M + H) m/z: calcd, 593.3114;
found 593.3107.
Kinetics of the Intramolecular Diels-Alder Reaction of
Adduct 6. Adduct 6 was purified by NP HPLC using 20% ethyl
acetate in hexanes. The peak corresponding to the acyclic adduct
was collected, and its purity was assessed by RP HPLC and NMR
spectroscopy. In order to assess the rate of cyclization from adduct
6 to 7 via IMDA reaction, a small volume of the work solution
was introduced into a much larger volume of solvent that had been
pre-equilibrated at the desired temperature, yielding a final 6
concentration of 50 µM. The conversion was followed by UV
spectroscopy in an Agilent 8453 photodiode-array spectrophotom-
eter in quartz cuvettes with Teflon stoppers. The temperature was
maintained at 37 °C by a circulating bath. The absorbance of the
solution was measured at 231 nm, the λmax of the conjugated diene,
every 2 min for 8 h. The decrease in absorbance at 231 nm was
fitted to a single-exponential decay.
Reduction of Adduct 6. To 100 µL aliquots of a 100 µM
solution of 6 in 10% isopropyl alcohol in ethyl acetate were added
a variety of reducing reagents. Thus, the following reagents were
examined in exploratory experiments: (A) 5 mg of CuCl crystals;
(B) 5 mg of Fe(NH4)2(SO4)2; (C) 10 mM thiourea in methanol;
(D) 5 mg of CuCl crystals and 8.4 M acetic acid; (E) 5 mg of
Fe(NH4)2(SO4)2 and 8.4 M acetic acid; (F) 5 mg of Zn dust and
8.4 M acetic acid;78 (G) 20 µM CuSO4 and 100 µM or 1 mM
ascorbic acid; (H) freshly scratched Mg turnings and iodine (in
methanol); (I) dimethyl sulfide at 1, 10, and 100 mM; (J) 100 mM
2-mercaptoethanol; (K) glutathione at 1 and 100 mM; (L) sodium
borohydride at 1 and 35 mM; and (M) 60 mM SnCl2. Each reaction
was left at room temperature for 18 h in the dark and then analyzed
by RP HPLC on a C18 column using solvent condition 3. The
reaction progress was monitored at 320 nm. The reaction mixtures
were compared with authentic standards of tyrosine analogue 4,
p-hydroxytyrosine analogue 11, and o-hydroxytyrosine analogue
12.
Oxidation of N-Acetyl-L-tyrosine Ethyl Ester (13) with
Methyl Linoleate. Methyl linoleate (118 mg) and 5 mg of 13 were
oxidized using 6 mg of MeOAMVN in 1 mL of 10% acetonitrile
in benzene. The reaction mixture was fractionated using NP HPLC.
The chromatograms of the adducts (Figure S10 in the Supporting
Information) were similar to those of the adducts formed in the
reaction of 4 and 5 but much more complex because of the
formation of regioisomers. Fraction A was collected, dried under
vacuum, resuspended in MeOH containing 0.1% acetic acid, and
analyzed by MS. The corresponding adducts were identified
[yielding (M + H) and (M + Na) peaks at m/z 575.93 and 598.27,
respectively] and further characterized by MS2 (Figure S11 in the
Supporting Information).
Synthesis of the Dipeptide N-Acetyl-L-tyrosine-L-alanine
Methyl Ester (14). To a flame-dried 10 mL round-bottom flask
were added N-acetyl-L-tyrosine (0.50 g, 2.24 mmol), L-alanine
methyl ester hydrochloride (0.31 g, 2.24 mmol), and 2-chloro-4,6-
dimethoxy-1,3,5-triazine (CDMT) (0.433 g, 2.46 mmol).79 The flask
was dried in vacuo for 20 min and then purged with argon.
Acetonitrile (2.5 mL) and then 4-methylmorpholine (NMM) (0.616
mL, 5.60 mmol) were added over 10 min, and the reaction was
stirred at 15 °C under an argon balloon for 2 h. The reaction was
quenched with H2O (12 mL) and extracted with ethyl acetate (25
mL). The organic layer was washed with 1 N HCl (2×, 6 mL),
H2O (12 mL), and brine (12 mL), dried over MgSO4, filtered, and
concentrated in vacuo. Silica flash column chromatography (10:90
MeOH/CH2Cl2) afforded the product, 14, as a white solid (0.50 g,
72.4%) upon solvent removal. 1H NMR (400 MHz, DMSO): δ 9.13
(s, 1H), 8.41 (d, J ) 7.0 Hz, 1H), 7.98 (d, J ) 8.5 Hz, 1H), 7.02
(d, J ) 8.5 Hz, 2H), 6.63 (d, J ) 8.5 Hz, 2H), 4.43 (td, J ) 9.8,
4.2 Hz, 1H), 4.26 (p, J ) 7.2 Hz, 1H), 3.60 (s, 3H), 2.85 (dd, J )
13.9, 4.2 Hz, 1H), 2.57 (dd, J ) 13.9, 10.1 Hz, 1H), 1.73 (s, 3H),
1.27 (d, J ) 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO): δ 173.31,
171.97, 169.42, 156.09, 130.42, 128.41, 115.18, 55.28, 54.28, 52.23,
47.91, 37.23, 22.83, 17.24. HRMS (M + Na) m/z: calcd, 331.1264;
found, 331.1255.
Synthesis of the p-Hydroxy Dipeptide N-Acetyl-L-p-hydro-
xytyrosine-L-alanine Methyl Ester (15). To a 10 mL round-bottom
flask were added PhI(OAc)2 (0.31 g, 0.973 mmol) and 20:80 H2O/
THF (3.0 mL) followed by dipeptide 14 (0.15 g, 0.486 mmol, in
3.0 mL of 20:80 H2O/THF) over 10 min. The reaction mixture
turned light-yellow upon initial dipeptide addition and then became
amber in color. The reaction mixture was stirred under atmosphere
at room temperature. Reaction progress was monitored via TLC
(10:90 MeOH/CH2Cl2) for loss of the starting dipeptide. After 2 h,
the reaction was deemed complete, and the solvent was removed
in vacuo, yielding an orange solid. Silica flash column chroma-
tography (5:95 MeOH/CH2Cl2) afforded the product, 15, as white
1
solid (0.11 g, 70%) upon solvent removal. H NMR (600 MHz,
Synthesis of o-Hydroxytyrosine Analogue 12. To a 100 mL
round-bottom flask under argon were added 3,4-dihydroxyhydro-
cinnamic acid (0.71 g, 3.90 mmol) and DMF (50 mL). To this
solution was added 4-bromomethyl-7-methoxycoumarin (0.91 g,
3.38 mmol) followed by triethylamine (0.57 mL, 3.72 mmol). The
reaction mixture turned light-yellow upon addition of the amine.
The flask was wrapped with aluminum foil and stirred at room
temperature under an argon balloon for 24 h. The solvent was
removed in vacuo, yielding the crude product as a brown oil. Silica
flash column chromatography (60:39:1 hexanes/EtOAc/TEA) af-
forded the product as a white solid (1.13 g, 90%) upon solvent
CDCl3): δ 7.08 (dd, J ) 10.4, 3.1 Hz, 1H), 6.97 (d, J ) 6.8 Hz,
1H), 6.91 (dd, J ) 10.3, 3.1 Hz, 1H), 6.51 (d, J ) 7.2 Hz, 1H),
6.24-6.16 (m, 2H), 4.73 (dd, J ) 13.3, 6.1 Hz, 1H), 4.54 (p, J )
7.2 Hz, 1H), 3.77 (s, 3H), 3.66 (s, 1H), 2.37 (dd, J ) 14.8, 5.6 Hz,
1H), 2.05 (s, 3H), 1.95 (dd, J ) 14.8, 6.3 Hz, 1H), 1.44 (d, J ) 7.2
Hz, 3H). 13C NMR (151 MHz, CDCl3): δ 184.78, 172.91, 170.68,
170.40, 149.86, 149.41, 128.24, 127.76, 68.15, 52.64, 49.12, 48.36,
43.56, 23.20, 17.97. HRMS (M + Na) m/z: calcd, 347.1214; found,
347.1210.
Synthesis of the o-Hydroxy Dipeptide N-Acetyl-L-o-hydroxy-
tyrosine-L-alanine Methyl Ester (16). To a 25 mL round-bottom
flask were added 3,4-dihydroxy-L-phenylalanine (0.50 g, 2.54
mmol) and deionized H2O (6.0 mL). The flask was purged with
argon, and acetic anhydride (Ac2O) (5.0 mL, 50.71 mmol) was
added over 1 h with an addition funnel. The reaction mixture
changed from milky-white to colorless upon Ac2O addition and
then became a slurry after 30 min. After 2 h at room temperature,
the solvent was removed in vacuo. Silica flash column chroma-
1
removal. H NMR (400 MHz, DMSO): δ 8.69 (s, 2H), 7.57 (d, J
) 8.9 Hz, 1H), 7.02 (d, J ) 2.5 Hz, 1H), 6.94 (dd, J ) 8.9, 2.5
Hz, 1H), 6.60 (m, 2H), 6.45 (dd, J ) 8.0, 2.1 Hz, 1H), 6.25 (s,
1H), 5.31 (d, J ) 1.0 Hz, 2H), 3.85 (s, 3H), 2.71 (s, 4H). 13C NMR
(101 MHz, DMSO): δ 172.31, 162.90, 160.30, 155.36, 150.71,
145.45, 143.94, 131.45, 126.23, 119.14, 116.04, 115.84, 112.70,
110.69, 109.66, 101.40, 61.59, 56.36, 35.62, 30.00. HRMS (M +
Na) m/z: calcd, 393.0945; found; 393.0938.
(78) Dai, P.; Dussault, P.; Trulinger, T. J. Org. Chem. 2004, 69, 2851.
(79) Smith, K. C.; White, R. L. J. Nat. Prod. 1995, 58, 1274.
9
J. AM. CHEM. SOC. VOL. 132, NO. 49, 2010 17499

A R T I C L E S
Shchepin et al.
tography (40:60 MeOH/CH2Cl2) afforded N-acetyl-3,4-dihydroxy-
chromatograms of the adducts were similar to those of the adducts
formed in the reaction of 4 and 5 but much more complex because
of the formation of regio- and stereoisomers (Figure S12 in the
Supporting Information). Fractions 6_1, 6_2, 7_3, and 7_4 were
collected from RP HPLC and analyzed by mass spectrometry. The
corresponding adducts yielding (M + H) and (M + Na) peaks at
m/z 633.08 and 649.75, respectively, were observed and further
characterized by MS2 (Figure S13 in the Supporting Information).
Fractions 6_1 and 7_4 were collected, and their 1D and 2 D NMR
spectra were acquired. A number of peroxyl-phenol coupled
adducts were formed, and the new downfield-shifted 13C signal in
the HMBC spectrum indicated the formation of para acyclic and
cyclic adducts, as demonstrated in the model system.
L-phenylalanine79 as a sticky white solid (0.30 g, 49%). H NMR
1
(300 MHz, D2O): δ 6.72 (d, J ) 8.0 Hz, 1H), 6.66 (s, 1H), 6.56
(d, J ) 8.1 Hz, 1H), 4.31-4.21 (m, 1H), 2.94 (dd, J ) 14.1, 4.7
Hz, 1H), 2.67 (dd, J ) 14.0, 8.6 Hz, 1H), 1.82 (s, 3H).
To a 10 mL round-bottom flask were added N-acetyl-3,4-
dihydroxy-L-phenylalanine (0.298 g, 1.25 mmol), L-alanine methyl
ester hydrochloride (0.17 g, 1.25 mmol), and CDMT (0.24 g, 1.37
mmol). The flask was dried in vacuo for 20 min and then directly
purged with argon. Acetonitrile (3.0 mL) was added, followed by
NMM (0.34 mL, 3.11 mmol) over 10 min.80 The reaction mixture
turned cloudy after the addition of NMM. The reaction mixture
was stirred at 15 °C under an argon balloon for 2 h, and then the
reaction mixture was quenched with H2O (12 mL) and extracted
with ethyl acetate (30 mL). The organic layer was washed with 1
N HCl (2×, 6 mL), H2O (12 mL), and brine (12 mL), dried over
MgSO4, filtered, and concentrated in vacuo. Silica flash column
chromatography (10:90 MeOH/CH2Cl2) afforded the product, 16,
as white solid (0.28 g, 70%) upon solvent removal. 1H NMR (600
MHz, D2O): δ 6.67 (d, J ) 8.1 Hz, 1H), 6.59 (d, J ) 1.7 Hz, 1H),
6.52 (dd, J ) 8.1, 1.7 Hz, 1H), 4.29 (t, J ) 7.7 Hz, 1H), 4.19 (q,
J ) 7.2 Hz, 1H), 3.50 (s, 3H), 2.73 (qd, J ) 13.8, 7.8 Hz, 2H),
1.79 (s, 3H), 1.15 (d, J ) 7.2 Hz, 3H). 13C NMR (151 MHz, D2O):
δ 174.36, 173.85, 172.80, 143.78, 142.78, 128.76, 121.46, 116.79,
116.07, 55.11, 52.80, 48.45, 36.35, 21.48, 16.12. HRMS (M + Na)
m/z: calcd, 347.1214; found, 347.1211.
Acknowledgment. We are grateful to Dr. Joy Joseph of the
Medical College of Wisconsin for a gift of BTBE. N.A.P.
acknowledges funding from the National Science Foundation and
support from the Center in Molecular Toxicology at Vanderbilt
University (T32 ES007028, Training Program in Environmental
Toxicology). Support from the Howard Hughes Medical Institute
to R.R., the National Institutes of Health to B.K. and R.R. (2
R01Hl063119-05), and Agencia Nacional de Investigacio´n e
Innovacio´n (ANII)/Fondo Clemente Estable to S.B. (FCE_362) is
also acknowledged. S.B. was partially supported by a fellowship
of ANII. R.R. is a Howard Hughes International Research Scholar.
Oxidation of Dipeptide 14 with Methyl Linoleate. Methyl
linoleate (400 mg) and 20 mg of dipeptide 14 were oxidized using
20 mg of MeOAMVN in 3.5 mL of 60% acetonitrile in benzene.
The reaction mixture was fractionated using NP HPLC. The
1
Supporting Information Available: Tables of H and 13C
NMR chemical shifts, 1D and 2D NMR spectra, HPLC-MS
data, experimental details, and complete ref 59. This material
is available free of charge via the Internet at http://pubs.acs.org.
(80) Garrett, C. E.; Jiang, X.; Prasad, K.; Repic, O. Tetrahedron Lett. 2002,
43, 4161.
JA106503A
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