Nitration reaction of lutein with peroxynitrite

Article abstract of DOI:10.1016/j.tetlet.2009.11.099

The in vitro reactivity of lutein toward peroxynitrite was investigated, and the reaction products produced by scavenging with peroxynitrite were analyzed. A novel lutein-6H-1,2-oxazine (1) along with 14-s-cis-15-nitirolutein (2) and 14′-s-cis-15′-nitrolutein (3) was isolated from the products of the reaction of lutein with peroxynitrite. These results indicate that lutein is able to capture peroxynitrite and nitrogen dioxide radicals from their molecules to form oxazine or nitrocarotenoids.

Full text of DOI:10.1016/j.tetlet.2009.11.099

Tetrahedron Letters 51 (2010) 676–678
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Tetrahedron Letters
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Nitration reaction of lutein with peroxynitrite
b
b
b
b
Makoto Tsuboi a, Hideo Etoh a,b, , Yuya Yomoda , Kyuki Kato , Hideaki Kato , Aditya Kulkarni ,
*
Yukimasa Terada c, Takashi Maoka d, , Hironobu Mori , Takahiro Inakuma
e
e
*
a Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
b Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
c Center for Computers and Technology, Meijo University, Shiogamaguchi, Tempaku, Nagoya 468-8502, Japan
d Research Institute for Production and Development, Shimogamo-morimotocho, Sakyo-ku, Kyoto 606-0805, Japan
e Research Institute, Kagome Co. Ltd, 17 Nishitomiyama, Nasushiobara-shi, Tochigi 329-2762, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The in vitro reactivity of lutein toward peroxynitrite was investigated, and the reaction products pro-
duced by scavenging with peroxynitrite were analyzed. A novel lutein-6H-1,2-oxazine (1) along with
14-s-cis-15-nitirolutein (2) and 140-s-cis-150-nitrolutein (3) was isolated from the products of the reac-
tion of lutein with peroxynitrite. These results indicate that lutein is able to capture peroxynitrite and
nitrogen dioxide radicals from their molecules to form oxazine or nitrocarotenoids.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 October 2009
Revised 17 November 2009
Accepted 24 November 2009
Available online 27 November 2009
Keywords:
Lutein
Reaction with peroxynitrite
Lutein-6H-1,2-oxazine
14-s-cis-15-Nitrolutein
140-s-cis-150-Nitrolutein
Peroxynitrite, the reaction product of superoxide and nitric
oxide, is a powerful oxidant produced by macrophages and neutro-
phils. Peroxynitrite is known to induce DNA strand scission,
protein modification by nitration, and hydroxylation and lipid per-
oxydation in LDL. Previously, we first reported the formation of
nitro-carotenoids by the reaction of b-carotene and astaxanthin
with peroxynitrite. These results indicated that b-carotene and
astaxanthin are able to capture peroxynitrite and nitrogen dioxide
radicals from molecules to form nitro-carotenoids.1,2 This informa-
tion would be of value to those investigating the peroxynitrite-
scavenging action of carotenoids in vivo.
a structure lutein NO adduct. The 1H and 13C NMR signals of 1 were
assigned by 1H–1H COSY, NOESY, HSQC, and HMBC experiments. The
13C NMR signals at C-5, C-6, C-7, and C-8 were significantly different
from those of lutein. The partial structure of C5-C6@C7-C8 was
elucidated from HMBC experiments. The chemical shift value of
the quaternary carbon at C-5 (d 80.1) indicated that an oxygen group
was attached to C-5. On the other hand, the chemical shift value of
the quaternary carbon at C-8 (d 142.6) indicated that nitrogen was
attached to C-8 by a double bond.7 These spectral data were in agree-
ment with the partial structure of –O-C5-C6@C7-C8@N–. From the
HRMS data, oxygen was found to be bound to nitrogen by a single
bond. Therefore, the partial structure of a six-membered oxazine
ring was elucidated. The remaining structural features were also
confirmed by NOESY correlations between CH3-16/17 and H-7,
CH3-19 and H-7/11, CH3-20 and H-11/15, CH3-160/170 and H-70,
CH3-190 and H-70/110, and CH3-200 and H-110/150. The HMBC spec-
trum showed cross peaks at CH3-16/17 to C-6, CH3-18 to C-5/6,
and CH3-19 to C-8/9/10, indicating a six-membered oxazine skele-
ton. Therefore, the structure of 1 was determined to be lutein-6H-
1,2-oxazine. The formation mechanism of 1 might be assumed to
be the direct reaction of lutein with peroxynitrite.
Lutein [(3R,30R,60R)-b, -carotene-3,30-diol] and its metabolites,
e
(3R,30S:meso)-zeaxanthin and 30-dehydrolutein, along with
(3R,30R)-zeaxanthin are presented in the macula and they perform
a defense function against oxidation injury in the eyes. Lutein also
prevents age-related macular degeneration (AMD).
In the present study, we investigated the reaction of lutein with
peroxynitrite because lutein has an asymmetric structure and so it
might provide some new reaction products by this reaction.
All-trans-lutein was reacted with peroxynitrite,3,4 and the reac-
tion products were analyzed by HPLC.
Compound 15,6 (yield 1.5 mg) showed absorption maxima at 430,
457, and 486 nm. Acetylation of 1 gave a diacetate. Its molecular for-
mula was determined to be C40H55O3N by HRFAB-MS, and it showed
18'
20
19
OH
3'
17 16
7
15
8
1'
H
C
1
15'
N
HMBC
16' 17'
HO
* Corresponding authors. Tel.: +81 75 781 1107; fax: +81 75 791 7659 (T.M.).
E-mail addresses: acheto@agr.shizuoka.ac.jp (H. Etoh), maoka@mbox.kyoto-
inet.or.jp (T. Maoka).
3
O
20'
19'
Lutein-6H-1,2-oxazine (1)
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.11.099

M. Tsuboi et al. / Tetrahedron Letters 51 (2010) 676–678
677
Compound 28 (yield 3.5 mg) showed absorption maxima at 322
and 465 nm. Its molecular formula was determined to be
C40H55O4N by HRFAB-MS and it demonstrated a NO2-substituted
lutein structure. This structure was also characterized from 1H
and 13C NMR including 2D NMR experiments. The partial structure
of the end group and the polyene chain of compound 2 were char-
acterized by 1H NMR and 13C NMR including 1H–1H COSY, NOESY,
HSQC, and HMBC experiments. The downfield shift of the 13C NMR
signal at C-15 (d 145.8, quaternary carbon) along with disappear-
ance of a methylene proton at the C-15 position in 1H NMR
compared with lutein, clearly indicated that a nitro group was at-
tached to the C-15 position of lutein. Furthermore, the change in
the coupling pattern and the downfield shifts of the 1H NMR
signals at H-150 (d 8.05) and H-14 (d 6.19) compared with lutein,
supported the substitution position of the nitro group at C-15.
The steric structure was confirmed by NOESY correlations between
CH3-16/17 and H-7, CH3-19 and H-7/11, CH3-20 and H-11/140, CH3-
160/170 and H-70, CH3-190 and H-70/110, and CH3-200 and H-110/150.
Spectral analysis of compound 2 indicated its structure to be 14-s-
cis-15-nitrolutein (2).
toward a wide variety of biological antioxidation systems would
enable us to predict the role of peroxynitrite in vivo and provide
valuable information on its physiological significance.
Acknowledgment
This work was supported in part by the Grant-in-Aid from the
Ministry of Education Science, Sports (Grant No. 20580126).
References and notes
1. Yoshioka, R.; Hayakawa, T.; Ishizuka, K.; Kulkarni, A.; Terada, Y.; Maoka, T.; Etoh,
H. Tetrahedron Lett. 2006, 47, 3637–3640.
2. Hayakawa, T.; Kulkarni, A.; Terada, Y.; Maoka, T.; Etoh, H. Biosci., Biotechnol.,
Biochem. 2008, 72, 2716–2722.
3. Niwa, T.; Doi, U.; Kato, Y.; Osawa, T. J. Agric. Food. Chem. 2001, 49, 177–182.
4. All-trans-lutein (400 mg, from Kemin Health Asia) was dissolved in 50 mL of THF
(final concentration 5.7 mM). To this, TFA was added in order to make up the
final concentration to 2%, before the addition of 16 mL of peroxynitrite (final
concentration: 6.8 mM). Then the solution was allowed to react for 1 min. Then,
to the above-mentioned mixture, 300 mL of CHCl3 and 300 mL of H2O were
added so as to separate the reaction products into organic and aqueous phases.
The whole procedure was performed for three times. The organic layer was
dried over sodium sulfate and concentrated. This organic concentrate was then
subjected to HPLC analysis using the Develosil C30-UG-5 (250 ꢀ 4.6 id; MeCN/
H2O = 82:18, flow rate: 1 min, column temp: 40 °C) column. A more specific
separation procedure was performed using the Deverosil C30-UG-5 (250 ꢀ 4.6
id mm; MeCN/H2O = 75:25) column.
3'
HO
1'
6'
19'
5. In HPLC analysis for lutein, four main groups of reaction products were
observed, namely fractions A (tR 3–12 min), B (tR 12–28 min), C (tR 30–36 min),
and D (tR 50–68 min). The peaks in fraction A were observed to have a lower
kmax, indicating then to be apo-carotenals. The fraction B and C compounds,
which contained the main reaction products, were observed to be oxygenated
products with a C-40 skeleton. The compounds in fraction D were 9- and 90-cis-
lutein and 13- and 130-cis-lutein. They were identified from their values in the
literature: Khachike, F.; Englert, G.; Daitch, C. E.; Beecher, G. R.; Lusby, W. R. J.
Chromatogr. Biomed. Appl. 1992, 582, 153–166. Further separation of fraction B
gave compounds 2 and 3 and fraction C gave compound 1.
20'
14'
20
19
15'
1
NO2
15
6
14
3
HO
6. Lutein-6H-1,2-oxazine (1) UV–vis kmax (Et2O) nm 430, 457, 486; HR-FAB MS
597.4173 (M+, calcd for C40H55O3N, 597.4182); 1H NMR (CDCl3, 500 MHz) d 0.85
(H3-170, s), 1.00 (H3-160, s), 1.23 (H-4
a, dd, J = 13.0, 4.0), 1.25 (H-2a, overlapped),
14-s-cis-15-nitrolutein (2)
1.26 (H3-17, s), 1.31 (H3-16, s), 1.37 (H-20a, dd, J = 13.0, 7.0), 1.60 (H3-18, s), 1.62
(H3-180, s), 1.85 (H-20b, dd, J = 13.0, 6.0), 1.91 (H3-190, s), 1.98 (H3-200, s), 1.99
(H3-20, s), 2.02 (H-2b, ddd, J = 13.0, 4.0, 2.0), 2.17 (H3-19, s), 2.41(H-60, d,
J = 10.0), 2.53 (H-4b, ddd, J = 13.0, 4.0, 2.0), 4.22 (H-3, m), 4.25 (H-30, m), 5.44 (H-
70, dd, J = 15.5, 10.0), 5.55 (H-40, br s), 6.14 (H-80, d, J = 15.5), 6.14 (H-100, d,
J = 11.0), 6.24 (H-7, s), 6.24 (H-12, d, J = 15.0), 6.25 (H-140, d, J = 11.0), 6.31 (H-14,
d, J = 11.0), 6.36 (H-120, d, J = 15.0), 6.62 (H-110, dd, J = 15.0, 11.0), 6.64 (H-15, m),
6.64 (H-150, m), 6.69 (H-11, dd, J = 15.0, 11.0), 8.30 (H-10, d, J = 11.0) 13C NMR
(CDCl3, 125 MHz) d 12.7 (C-20), 12.8 (C-200), 13.1 (C-190), 15.1 (C-19), 22.9 (C-
180), 23.4 (C-18), 24.2 (C-170), 26.5 (C-17), 29.4 (C-16), 29.5 (C-160), 34.0 (C-10),
34.8 (C-1), 44.6 (C-20), 45.9 (C-4), 51.4 (C-2), 54.9 (C-60), 65.6 (C-3), 65.9 (C-30),
80.1 (C-5), 116.1 (C-7), 124.5 (C-40), 125.1 (C-110), 128.3 (C-9), 128.8 (C-70),
129.8 (C-15), 129.9 (C-11), 130.7 (C-150), 130.8 (C-100), 132.4 (C-12), 132.5 (C-
13, 130), 134.1 (C-10), 134.5 (C-14), 135.3 (C-90), 137.1 (C-120, 140), 137.7 (C-80),
138.0 (C-50), 142.6 (C-8), 156.3 (C-6); acetylation of 1 with acetic anhydride in
pyridine at room temperature for 1 h gave a diacetate, which showed molecular
ion m/z 681 by FAB MS.
Compound 39 (yield 3.2 mg) showed maxima at 343 and
447 nm and molecular formula as those of 2. The 1H and 13C
NMR data of 3 were very similar to those of 2 except for at the
14, 15, 140, and 150 positions. The quaternary carbon at C-150 (d
145.8) and doublet signal at H-15 (d 8.06) clearly indicated that a
nitro group was attached to C-150. Its steric structure was con-
firmed by NOESY data. The final structure of compound 3 was
established as 140-s-cis-150-nitrolutein (3).
19
20
15
14
20'
NO2
15'
1
6
3
HO
14'
7. Kalinowski, H.-O.; Berger, S.; Braun, S. Carbon-13 NMR spectroscopy; John Wiley
& Son Ltd: New York, 1988. p. 243 and 391.
8. 14-s-cis-15-Nitrolutein (2) UV–vis kmax (Et2O) nm 322, 465; HR-FAB MS
613.4139 (M+, calcd for C40H55O4N, 613.4131); 1H NMR (CDCl3, 500 MHz) d
0.85 (H3-160, s), 1.00 (H3-170, s) 1.09 (H3-16 and 17, 2s), 1.37 (H-20a, dd, J = 14.0,
19'
1'
7.0), 1.48 (H-2
s), 1.77 (H-2b, ddd, J = 12.0, 4.0, 1.5), 1.84 (H-20b, dd, J = 14.0, 6.0), 1.95 (H3-190,
s), 1.99 (H3-19, s), 2.04 (H-4
, dd, J = 18.0, 10.0), 2.16 (H3-200, s), 2.40 (H-4b, ddd,
a
, dd, J = 12.0, 11.0), 1.62 (H3-180, s), 1.75 (H3-18, s), 1.77 (H3-20,
6'
a
J = 18.0, 6.0, 1.5), 2.43 (H-60, d, J = 10.0), 4.00 (H-3, m), 4.25 (H-30, m), 5.56 (H-40,
s), 5.56 (H-70, dd, J = 15.0, 10.0), 5.86 (H-140, d, J = 11.0), 6.08 (H-10, d, 11.5), 6.08
(H-100, d, J = 11.0), 6.10 (H-7, d, J = 16.0), 6.16 (H-8, d, J = 16.0), 6.16 (H-80, d,
J = 15.0), 6.19 (H-14, s), 6.40 (H-120, d, J = 15.0), 6.50 (H-12, d, J = 15.0), 6.79 (H-
11, dd, J = 15.0, 11.5), 6.90 (H-110, dd, J = 15.0, 11.0), 8.05 (H-150, d, J = 12.0) 13C
NMR (CDCl3, 125 MHz) d 13.1 (C-19), 13.3 (C-190), 13.6 (C-200), 15.3 (C-20), 20.5
(C-17), 22.9 (C-18, 180), 24.3 (C-170), 28.7 (C-16), 29.5 (C-160), 34.0 (C-10), 37.1
(C-1), 42.6 (C-4), 44.6 (C-20), 48.4 (C-2), 54.9 (C-60), 65.0 (C-3), 65.9 (C-30), 118.9
(C-14), 124.6 (C-40), 125.6 (C-140), 126.8 (C-5, 7), 127.9 (C-11), 130.0(C-100),
130.4 (C-110, 150), 131.1 (C-70), 131.2 (C-10), 135.7 (C-12), 136.1 (C-90, 120),
137.6 (C-50, 6, 80), 138.1 (C-9), 138.2 (C-8), 142.8 (C-13), 145.8 (C-15), 149.1 (C-
130).
3'
OH
14'-s-cis-15'-nitrolutein (3)
The versatility of the reaction mode is suggestive of the involve-
ment of several different active species in the reaction with perox-
ynitrite. There are still many unidentified products, the
identification of which may provide additional new reaction modes
for the reaction of peroxynitrite with carotenoids as well as various
other biological antioxidation systems. These reactions would
probably be found in vivo and contribute to the degradation of bio-
logical systems, eventually leading to pathogenic disease pro-
cesses. Better understanding of the behavior of peroxynitrite
9. 140-s-cis-150-Nitrolutein (3) UV–vis kmax (Et2O) nm 343, 447; HR-FAB MS
613.4139 (M+, calcd for C40H55O4N, 613.4131); 1H NMR (CDCl3, 500 MHz) d
0.86 (H3-160, s), 1.10 (H3-16, s) 1.08 (H3-16 and 17 , 2s), 1.37 (H-20a, dd, J = 14.0,
7.0), 1.48 (H-2
a
, dd, J = 12.0, 11.0), 1.64 (H3-180, s), 1.74 (H3-18, s), 1.76 (H3-200,
s), 1.77 (H-2b, ddd, J = 12.0, 4.0, 1.5), 1.85 (H-20b, dd, J = 14.0, 6.0), 1.93 (H3-190,

678
s), 2.01 (H3-19, s), 2.04 (H-4
M. Tsuboi et al. / Tetrahedron Letters 51 (2010) 676–678
, dd, J = 18.0, 10.0), 2.16 (H3-20, s), 2.40 (H-4b, ddd,
a
21.6 (C-18), 22.9 (C-180), 24.3 (C-170), 28.7 (C-16), 29.5 (C-160), 34.0 (C-10), 37.1
(C-1), 42.6 (C-4), 44.6 (C-20), 48.4 (C-2), 54.9 (C-60), 65.0 (C-3), 65.9 (C-30), 119.0
(C-140), 124.6 (C-40), 125.8 (C-14), 126.8 (C-5, 7), 127.8 (C-110, 120), 130.1 (C-70,
100), 130.2 (C-10), 130.4 (C-11, 15), 136.1 (C-9, 12), 137.6 (C-50, 6, 80), 138.0 (C-
8), 138.1 (C-90), 142.8 (C-130), 145.8 (C-150), 149.1 (C-13); NOESY correlations
between CH3-16/17 and H-7, CH3-19 and H-7/11, CH3-20 and H-11/15, CH3-160/
170 and H-70, CH3-190 and H-70/110, and CH3-200 and H-14/110.
J = 18.0, 6.0, 1.5), 2.43 (H-60, d, J = 10.0), 4.00 (H-3, m), 4.25 (H-30, m), 5.40 (H-70,
dd, J = 15.0, 10.0), 5.56 (H-40, s), 5.96 (H-14, d, J = 12.0), 6.10 (H-100, d, J = 11.0),
6.10 (H-7, d, J = 16.0), 6.16 (H-8, d, J = 16.0), 6.16 (H-10, d, J = 11.5), 6.20 (H-80, d,
J = 15.0), 6.30 (H-140, s), 6.40 (H-12, d, J = 15.0), 6.49 (H-120, d, J = 15.0), 6.75 (H-
110, dd, J = 15.0, 11.0), 6.95 (H-11, dd, J = 15.0, 11.5), 8.06 (H-15, d, J = 12.0) 13C
NMR (CDCl3, 125 MHz) d 13.2 (C-19, 190), 13.6 (C-20), 15.3 (C-200), 20.5 (C-17),