Novel carotenoid pyropheophorbide A esters from abalone

Article abstract of DOI:10.1016/j.tetlet.2011.03.146

A series of carotenoid pyropheophorbide A esters, fucoxanthin pyropheophorbide A ester (1), halocynthiaxanthin 3′-acetate pyropheophorbide A ester (2), lutein pyropheophorbide A esters (3) and (4), and mutatoxanthin pyropheophorbide A ester (5), were isolated from the viscera of the abalone Haliotis diversicolor aquatilis. These structures were determined based on UV-vis, MS, and NMR spectroscopic data.

Full text of DOI:10.1016/j.tetlet.2011.03.146

Tetrahedron Letters 52 (2011) 3012–3015
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Tetrahedron Letters
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Novel carotenoid pyropheophorbide A esters from abalone
Takashi Maoka a, , Tetsuji Etoh b, Naoshige Akimoto b, Hiroyuki Yasui c
⇑
a Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto 606-0805, Japan
b Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
c Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of carotenoid pyropheophorbide A esters, fucoxanthin pyropheophorbide A ester (1), halocyn-
thiaxanthin 30-acetate pyropheophorbide A ester (2), lutein pyropheophorbide A esters (3) and (4), and
mutatoxanthin pyropheophorbide A ester (5), were isolated from the viscera of the abalone Haliotis diver-
sicolor aquatilis. These structures were determined based on UV–vis, MS, and NMR spectroscopic data.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 March 2011
Revised 28 March 2011
Accepted 31 March 2011
Available online 7 April 2011
Since the first structural elucidation of b-carotene by Kuhn and
Karrer in 1928–1930, about 750 naturally occurring carotenoids
had been reported as of 2004.1 Marine animals contain various
carotenoids which show structural diversity.2,3 Still interesting
structural carotenoids can be found in marine animals.2,3
In the course of our studies on carotenoids in marine animals,3
novel carotenoid pyropheophorbide A esters (Fig. 1) were isolated
from the viscera of the abalone Haliotis diversicolor aquatilis. This
Letter reports the isolation and structural elucidation of these
compounds.
and pyropheophorbide A,6 are compiled in Tables 1 and 2. NMR
data revealed that the pyropheophorbide A and fucoxanthin moie-
ties were linked with esterified bond. The 1H NMR signal of H-3
(4.82 ppm) in the fucoxanthin moiety in 1, which showed about
1 ppm downfield shift relative to the corresponding signal in fuco-
xanthin,7 clearly indicated that the hydroxy group at C-3 in the
fucoxanthin moiety was esterified with pyropheophorbide A. The
key ROESY and HMBC correlations are shown in Figure 2. There-
fore, the structure of brown pigment 1 was determined as fucoxan-
thin 3-pyropheophorbides A ester, as shown in Figure 1.
Viscera of abalone (100 g from 50 specimens) were extracted
with Me2CO. The Me2CO extract was partitioned with hexane/
Et2O (1:1) and water. The hexane/Et2O (1:1) layer was evaporated
to dryness and chromatographed on silica gel using an increasing
percentage of Et2O in hexane. The fraction eluted with Et2O/hexane
(6:4) was subjected to HPLC on silica gel with Me2CO/hexane (3:7)
to yield a series of brown pigments: 1 (6.6 mg), 2 (1.0 mg), 3
(0.2 mg), 4 (0.2 mg), and 5 (0.2 mg).
Compound 1 showed absorption maxima at 226, 317, 411, 447,
469, 538, 608, and 666 nm.4 This UV–vis spectrum resembled the
additive UV–vis spectra of fucoxanthin5 and pyropheophorbide
A.6 The molecular formula of 1 was determined as C75H90O8N4 by
high-resolution (HR) FAB-MS.4 The characteristic fragment ion at
m/z 535.2719 corresponded to the pseudomolecularion (MH+) of
pyropheophorbide A (C33H35O3N4), suggesting the presence of a
pyropheophorbide A moiety in 1 (Fig. 2). These data indicated that
compound 1 consisted of fucoxanthin and pyropheophorbide A
moieties. Both fucoxanthin and pyropheophorbide A structural
moieties in 1 were characterized by the 1H NMR and 13C NMR spec-
tra, including 2D NMR (COSY, ROESY, HSQC, and HMBC) experi-
ments. NMR spectral data of 1, assigned on the basis of the
results of 2D NMR experiments and comparison with fucoxanthin7
Compound 2 showed almost the same absorption spectrum as
1.8 The molecular formula of 2 was determined as C75H88O7N4 by
HRFAB-MS.8 The characteristic fragment ion at m/z 535 was also
observed, indicating the presence of a pyropheophorbide A moiety.
The 1H NMR and 13C NMR spectra (Tables 1 and 2) of 2, assigned
based on 2D NMR experiments, showed that compound 2 con-
sisted of halocynthiaxanthin 30-acetate7 and pheophorbide A moi-
eties with esterified linkage as shown in Figure 1. Therefore, the
structure of 2 was determined as halocynthiaxanthin 30-acetate
pheophorbide A ester.
Both compounds 3 and 4 showed the same UV–vis spectra.9,10
The molecular formula of both compounds was determined as
C
73H88O4N4 by HRFAB-MS.9,10 The characteristic fragment ion at
m/z 535 was also observed in both compounds. The 1H NMR data
(Tables 1 and 2) indicated the presence of lutein7 and pheophor-
bide A moieties both in 3 and 4. Detailed 1H NMR analysis includ-
ing decoupling, COSY and ROESY experiments revealed that the
hydroxy group at C-3 at the b-end group of lutein was esterified
with pheophorbide A in the case of 311 and hydroxy group at the
e
-end group in lutein was esterified with pheophorbide A in the
case of 4,12 as shown in Figure 1.
Compound 5 showed absorption maxima at 226, 317, 411, 428,
457, 538, 608, and 666.13 The molecular formula of 5 was deter-
mined as C73H88O5N4 by HRFAB-MS.13 As well as in other com-
pounds, the characteristic fragment ion at m/z 535 was also
observed. The 1H NMR (Tables 1 and 2) of 5 showed that compound
⇑
Corresponding author. Tel.: +81 75 781 1107; fax: +81 75 781 1118.
E-mail address: maoka@mbox.kyoto-inet.or.jp (T. Maoka).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.146

T. Maoka et al. / Tetrahedron Letters 52 (2011) 3012–3015
3013
OH
121"
12"
3'
82"
O
81"
8"
5'
19
20
10"
11"
7'
.
O
1'
9"
13"
10'
15
15'
7"
6"
71"
O
17 16O
131"
14"
16' 17'
HN
N
10
132"
7
15"
20'
19'
5"
O
1
4"
1
173"
O
16"
17"
O
17 "
N
NH
1"
5
3
3"
19"
31"
172"
18
1
O
2"
21"
18"
20"
32"
181"
O
O
O
a
O
O
OH
OH
O
HN
N
O
b
c
N
NH
R
O
O
2: R=a
3: R=b
4: R=c
5: R=d
OH
O
O
d
Figure 1. Structures of compounds 1–5.
OH
O
O
.
O
O
HN
N
O
O
N
NH
O
1
H
C
534+H m/z 535
ROESY correlation
HMBC correlation
Figure 2. Key ROESY and HMBC correlations and the fragment ion of 1.
Table 1
1H NMR (500 MHz) and 13C NMR (125 MHz) data of carotenoid parts of compounds 1 and 2 and 1H NMR (500 MHz) of compounds 3–5 in CDCl3
Position
1
2
3
4
5
d 13C
d1H
mult. J (Hz)
d 13C
d1H
mult. J (Hz)
d1H
mult. J (Hz)
d1H
mult. J (Hz)
d1H
mult. J (Hz)
1
2
2
3
4
4
35.1
42.8
35.1
42.8
a
1.37
Overlapped
dd (13, 12)
m
Overlapped
Overlapped
a
1.37
Overlapped
dd (13, 12)
m
Overlapped
Overlapped
a
1.60
Overlapped
Overlapped
m
Overlapped
Overlapped
a
1.36
dd (14,7)
dd (14, 6)
m
a
1.60
Overlapped
Overlapped
m
Overlapped
Overlapped
b 1.21
4.82
a
b 1.21
4.82
a
b 1.39
5.00
a
b 1.77
5.26
5.34
b 1.39
5.00
a
68.0
37.6
68.0
37.6
2.27
2.27
2.53
br s
2.53
b 1.74
b 1.74
b 1.98
b 1.98
5
6
7
7
67.0
65.8
40.6
67.0
65.8
40.6
2.34
5.38
d (10)
dd (15, 10)
2.56
3.60
d (17)
d (17)
2.56
3.60
d (17)
d (17)
6.03
6.09
d (18)
d (18)
6.03
6.09
d (18)
d (18)
8
9
10
11
197.5
134.4
139.0
125.6
197.5
134.4
139.0
125.6
6.09
d (15)
7.10
6.45
d (11.5)
dd (15, 11.5)
7.10
6.45
d (11.5)
dd (15, 11.5)
6.14
ꢀ6.62
d (11)
Overlapped
6.14
ꢀ6.62
d (10)
Overlapped
6.14
ꢀ6.62
d (11)
Overlapped
(continued on next page)

3014
T. Maoka et al. / Tetrahedron Letters 52 (2011) 3012–3015
Table 1 (continued)
Position
1
2
3
4
5
d 13C
d1H
mult. J (Hz)
d 13C
d1H
mult. J (Hz)
d1H
mult. J (Hz)
d1H
mult. J (Hz)
d1H
mult. J (Hz)
12
13
14
15
16
17
18
19
20
149.0
136.1
136.6
129.4
24.6
27.6
21.4
11.7
13.0
6.63
d (15)
149.0
136.1
136.6
129.4
24.6
27.6
21.4
11.7
13.0
6.63
d (15)
6.36
d (15)
6.37
d (15)
6.36
d (15)
6.40
6.60
0.99
0.83
1.14
1.89
1.98
d (11)
dd (14, 11)
6.40
6.60
0.99
0.83
1.14
1.89
1.98
d (11)
dd (14, 11)
6.25m
ꢀ6.62
1.03
0.96
1.65
6.25
ꢀ6.62
1.07
0.80
1.60
1.88
1.96
m
6.25m
ꢀ6.62
1.03
0.96
1.65
Overlapped
Overlapped
Overlapped
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
1.94
1.96
1.94
1.96
10
35.7
45.2
36.1
42.3
20
a
1.99
Overlapped
dd (12.5, 7)
a
1.83
ddd (12, 3, 1.5)
dd (12, 12)
m
ddd(18,5,1.5)
dd (18, 10)
a
1.37
dd (14,7)
dd (14, 6)
m
a
1.77
dd(12, 3)
dd (12, 12)
m
Overlapped
dd (18, 10)
a
1.51
Overlapped
Overlapped
m
Overlapped
Overlapped
20
b 1.41
b 1.57
5.04
b 1.85
4.25
5.55
b 1.48
4.00
b 1.76
4.25
30
67.9
45.4
68.0
37.50
40
a
2.27
Overlapped
dd (13, 12.5)
a
2.49
br s
a
2.39
a
2.12
40
b 1.51
b 2.13
b 2.04
b 1.99
50
72.7
137.0
124.3
88.8
60
117.5
202.3
103.3
132.5
128.3
125.6
137.8
139.0
131.6
132.1
29.2
32.1
31.3
14.0
12.9
170.4
20.8
2.41
5.46
6.14
d (10)
dd (15, 10)
d (15)
70
6.10
6.15
d (16)
d (16)
5.23
5.17
s
s
80
6.05
s
98.7
90
119.3
135.1
124.3
138.0
136.6
135.5
130.7
28.7
30.2
22.4
13.0
18.6
100
110
120
130
140
150
160
170
180
190
200
3-COCH3
3-COCH3
6.31
6.64
6.35
d (11.5)
dd (15, 11.5)
d (15)
6.45
6.52
6.37
d (11)
dd (15, 11)
d (15)
6.14
d (11)
Overlapped
d (15)
6.15
d (11)
Overlapped
d (15)
6.19
6.59
d (11)
dd (15, 11)
ꢀ6.62
ꢀ6.64
6.37
6.36
6.26
6.74
1.38
1.07
1.35
1.81
1.98
d (11)
dd (14, 11)
6.29
6.64
1.18
1.20
1.92
2.01
1.97
m
m
s
s
s
6.25
ꢀ6.62
1.00
0.85
1.63
1.92
1.99
m
6.25
ꢀ6.62
1.07
1.07
1.74
1.97
1.97
m
Overlapped
Overlapped
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
1.17
1.33
1.62
1.71
1.94
s
s
s
s
s
s
s
170.7
21.6
2.04
s
2.04
s
s: Singlet, d: doublet, t triplet, m: multiplet, br s: broad singlet.
Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) data of pyropheophorbide A part of compounds 1 and 2 and 1H NMR (500 MHz) of compounds 3–5 in CDCl3
Position
d
13C
d1H
mult. J (Hz)
Position
1200
d
13C
d1H
mult. J (Hz)
100
200
141.5
132.4
12.7
136.1or 137.8
128.3
128.5 or 130.5
12.5
128.5 or 130.5
193.6
121
H3 3.68
s
00
21
H3 3.42
s
1300
00
300
131
00
00
00
31
8.00
6.29
6.18
dd (17, 11.5)
d (17)
d (11.5)
132
48.1
5.27
5.11
d (20)
d (20)
00
00
00
32
125.5
132
32
1400
1500
1600
1700
106.4 or 150.7
106.4 or150.7
160.4
51.5
29.7
400
500
600
700
136.1 or 137.8
97.1
137.1
155.2
12.1
144.9
19.5
17.5
9.41
s
s
4.31
2.34
2.70
2.20
2.48
m
m
m
m
m
171
00
00
00
00
71
H3 3.26
171
00
800
172
31.3
81
H3 3.71
H3 1.70
q (7.5)
t (7.5)
172
00
00
82
173
171.8
49.9
23.2
172.5
93.0
00
900
138.3
104.1
139.0
1800
4.50
1.81
m
d (6)
1000
1100
9.51
s
181
00
1900
2000
8.57
s
s: Singlet, d: doublet, t: triplet, m: multiplet.
5 consisted of mutatoxanthin7 and pheophorbide A moieties with
esterified linkage as shown in Figure 1.
pyropheophorbide A by esterase in the abalone viscera. It is well-
known that pyropheophorbide A is a photosensitizer and generates
singlet oxygen (1O2) from ground-state molecular oxygen in the
presence of light.16 On the other hand, carotenoids are excellent
quenchers of 1O2 and prevent photooxidation.17 Therefore, it is
interesting that compounds acting as singlet oxygen generators
and quenchers are linked with esterified bonds.
The major food sources of abalone are macroalgae such as
brown and red algae, which contain fucoxanthin and lutein as ma-
jor carotenoids, respectively.14 Abalone accumulate fucoxanthin
and lutein in the viscera from dietary algae. Halocynthiaxanthin
30-acetate is a metabolite of fucoxanthin in shellfish.2,3 Mutaoxan-
thin is derived from antherxanthin under acidic conditions.15 Pyr-
opheophorbide A is a metabolite of chlorophyll A in the viscera of
abalone.16 Compounds 1–5 might be formed from carotenoids and
To investigate whether 1O2 is generated from a porphyrin (pyr-
opheophorbide A), and whether the compounds such as an 1O2
quencher (fucoxanthin) and carotenoid pyropheophorbide A ester

T. Maoka et al. / Tetrahedron Letters 52 (2011) 3012–3015
3015
(a) Pyropheophorbide A: relative ESR signal intensity = 2.96
(b) Pyropheophorbide A + Fucoxanthin: relative ESR signal intensity = 0.50
(c) Fucoxanthin pyropheophorbide A ester: relative ESR signal intensity = 1.85
(natural product found in abalone)
Figure 3. ESR spectra due to the singlet oxygen adducts of TMPD (4-oxo-TEMPO) derived from (a) pyropheophorbide A, (b) pyropheophorbide A and fucoxanthin, and (c) 1 at
30 s after UVA exposure.
(1) react with 1O2 chemically, we examined the 1O2 quenching
10. Compound 4: UV–vis (Et2O) nm 226, 317, 411, 445, 474, 538, 608, 666; HRFAB-
MS m/z 1085.6867 (MH+) C73H89O4N4, calcd for 1085.6884, 1H NMR (Tables 1
activities of free and conjugated fucoxanthin in the chemical sys-
and 2).
tems employing the ESR spin-trapping method.18 1O2 was gener-
11. The esterified oxy methine signal at C-3 in 3 was observed at 5.00 ppm, which
was assigned by oxy methine signal at C-3 in the b-end group in lutein by COSY
ated in the pyropheophorbide A solution under UVA irradiation.
The signal intensity due to 4-oxo-TEMPO (2.96) decreased follow-
ing additions of free (0.50) and conjugated (1.85) fucoxanthin in a
structure-dependent manner, indicating that free fucoxanthin re-
acted chemically with 1O2. However, fucoxanthin conjugated with
pyropheophorbide A had a partial effect, as shown in Figure 3.
and ROESY experiments and showed about 1 ppm downfield shift relative to
the corresponding signal in lutein.7 This clearly indicated that the hydroxy
group at C-3 in the b-end group in lutein moiety was esterified with
pyropheophorbide A in the case of 3. Key COSY correlations H-2
a/H-3, H-2b/
H-3, H-4
H-3.
a/H-3, H-4b/H-3; key ROESY correlations H-2
a
/H-3, H-4
a/H-3, H-16/
12. The esterified oxy methine signal at C-3 in 4 was observed at 5.26 ppm, which
was assigned by oxy methine signal at C-3 in the -end group in lutein by COSY
e
and ROESY experiments and showed about 1 ppm downfield shift relative to
References and notes
the corresponding signal in lutein.7 This clearly indicated that the hydroxy
group at C-3 in the
pyropheophorbide A in the case of 4. Key COSY correlations H-2
H-3, H-4/H-3; key ROESY correlations H-2 /H-3, H-16/H-3.
e
-end group in lutein moiety was esterified with
1. Carotenoids Hand Book; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.;
Birkhäuser: Basel, Switzerland, 2004.
2. Matsuno, T. Fisheries Sci. 2001, 67, 771–789.
3. Maoka, T. Arch. Biochem. Biophys. 2009, 483, 191–195.
4. Compound 1: UV–vis (Et2O) nm (e) 226 (20,000), 317 (18,000), 411 (84,000),
a/H-3, H-2b/
a
13. Compound 5: UV–vis (Et2O) nm 226, 317, 411, 428, 457, 538, 608, 666; HRFAB-
MS m/z 1101.6848 (MH+) C73H89O5N4, calcd for 1101.6833, 1H NMR (Tables 1
and 2); key COSY correlations H-2
a/H-3, H-2b/H-3, H-4a/H-3, H-4b/H-3; key
447 (63,000), 469 shoulder (53,000), 538 (7000), 608 (5000), 666 (30,000);
HRFAB-MS m/z 1175.6827 (MH+) C75H91O8N4, calcd for 1175.6837, fragment
ion at m/z 535.2719 C33H35O3N4, calcd for 535.2709; 1H NMR and 13C NMR
ROESY correlations H-2 /H-3, H-4
a
a/H-3, H-16/H-3. The esterified oxy methine
signal at C-3 in 5 was observed at 5.00 ppm, which was assigned by oxy
methine signal at C-3 in the b-end group in mutatoxanthin by COSY and ROESY
experiments.
(Tables 1 and 2); CD (Et2O) nm (De), 240 (ꢁ1.0), 265 (ꢁ0.8), 296 (ꢁ3.2), 320
(ꢁ0.2), 340 (ꢁ1.5).
14. Liaaen-Jensen, S. Carotenoids in chemosystematics. In Carotenoids; Britton, G.,
Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser: Basel, 1995; pp 217–247. Vol. 3.
15. Eugster, C. H. Derivatization Microscale tests for the presence of common
functional groups in carotenoids. In Carotenoids; Britton, G., Liaaen-Jensen, S.,
Pfander, H., Eds.; Birkhäuser: Basel, 1995; pp 71–80. Vol. 1A.
5. Britton, G. UV/Visible Spectroscopy. In Carotenoids; Britton, G., Liaaen-Jensen,
S., Pfander, H., Eds.; Birkhäuser: Basel, 1995; pp 13–62. Vol. 1B.
6. Kobayashi, M.; Kanda, F.; Kamiya, H. Nippon Suisan Gakkaishi 1991, 57, 1983.
7. Englert, G. NMR Spectroscopy In Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.;
Birkhäuser: Basel, 1995; pp 147–260. Vol. 1B.
16. Tstumi, J.; Hashimoto, Y. Agric. Biol. Chem. 1964, 28, 467–470.
17. Foote, C. S.; Denny, R. W. J. Am. Chem. Soc. 1968, 90, 6233–6235.
8. Compound 2: UV–vis (Et2O) nm (e) 226 (20,000), 317 (18,000), 411 (84,000),
447 (63,000), 469 shoulder (53,000), 538 (7,000), 608 (5,000), 666 (30,000);
HRFAB-MS m/z 1157.6709 (MH+) C75H89O7N4, calcd for 1157.6731, fragment
ion at m/z 535.2730 C33H35O3N4, calcd for 535.2709; 1H NMR and 13C NMR
18. In Figure 3, the reaction mixture contained 50 mM TMPD, (a) 62.5
pyropheophorbide A, (b) 62.5 pyropheophorbide and 62.5
fucoxanthin, and (c) 62.5
phosphate buffer (pH 7.5) and ethanol (1:1, v/v).
l
l
M
M
l
M
A
l
M 1 in a total volume of 0.2 mL of 50 mM
(Tables 1 and 2); CD (Et2O) nm (De),240 (ꢁ1.0), 265 (ꢁ0.8), 296 (ꢁ3.2), 320
(ꢁ0.2), 340 (ꢁ1.5).
9. Compound 3: UV–vis (Et2O) nm 226, 317, 411, 445, 474, 538, 608, 666; HRFAB-
MS m/z 1085.6864 (MH+) C73H89O4N4, calcd for 1085.6884, 1H NMR (Tables 1
and 2).