Terreusinone, a novel UV-A protecting dipyrroloquinone from the marine algicolous fungus Aspergillus terreus

Article abstract of DOI:10.1016/j.tetlet.2003.08.101

A novel chiral dipyrrolobenzoquinone derivative, terreusinone (1), has been isolated as a potent UV-A protectant from the marine algicolous fungus Aspergillus terreus. The structure and absolute stereochemistry of the new compound was established by spectral interpretation, Horeau's method and quantum chemistry calculations as 2,6-bis[(1R)-1-hydroxyisobutyl]-1H,5H-pyrrolo[2,3-b]indole-4,8-dione (1). Compound 1 exhibited a UV-A absorbing activity with ED₅₀ value of 70 μg/mL.

Full text of DOI:10.1016/j.tetlet.2003.08.101

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 7707–7710
Terreusinone, a novel UV-A protecting dipyrroloquinone from
the marine algicolous fungus Aspergillus terreus
Sang Mi Lee,a Xi Feng Li,a Hualiang Jiang,b Jia Gao Cheng,b Seeyearl Seong,c Hong Dae Choid and
Byeng Wha Sona,*
aDepartment of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea
bDepartment of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031,
People’s Republic of China
cComputer Aided Molecular Design Research Center, Soongsil University, Seoul 156-743, Republic of Korea
dDepartment of Chemistry, Dongeui University, Busan 614-714, Republic of Korea
Received 6 August 2003; accepted 28 August 2003
Abstract—A novel chiral dipyrrolobenzoquinone derivative, terreusinone (1), has been isolated as a potent UV-A protectant from
the marine algicolous fungus Aspergillus terreus. The structure and absolute stereochemistry of the new compound was established
by spectral interpretation, Horeau’s method and quantum chemistry calculations as 2,6-bis[(1R)-1-hydroxyisobutyl]-1H,5H-
pyrrolo[2,3-b]indole-4,8-dione (1). Compound 1 exhibited a UV-A absorbing activity with ED50 value of 70 mg/mL.
© 2003 Elsevier Ltd. All rights reserved.
Marine microorganisms have proven to be a rich source
of structurally novel and biologically active natural
products required for developing fine chemical
agents.1,2
absorbing activity and was separated by assay-guided
fractionation using repeated silica gel flash chromato-
graphy (n-hexane in ethyl acetate) and HPLC (ODS-A,
MeOH–H2O=5:1) to yield a new dipyrroloquinone,
terreusinone (1, 18 mg).
In the course of our search for UV-A absorbing con-
stituents produced by fungi isolated from marine habi-
tats,3 moderate UV-A absorbing activity was detected
in the crude extract of the algicolous fungus Aspergillus
terreus. Here, we report on the structural elucidation of
the dipyrroloquinone, terreusinone (1), isolated as the
target compound of activity from the marine algicolous
fungus Aspergillus terreus.
The fungal strain (culture c MFA 460) was isolated
from the surface of the marine red alga Halymenia
acuminata collected in the Bijin Island, Gyeongnam
Province in 2002. The fungus, identified to be an Asper-
gillus terreus by fatty acid methyl ester analysis,4 was
cultured (10 L) in a seawater-based medium.5 The
resulting broth and mycelium were extracted separately
to afford crude extracts of 1.0 g and 8.0 g, respectively.
The broth extract (EtOAc) revealed a moderate UV-A
Terreusinone (1)6 was isolated as a yellowish solid
which analyzed for C18H22N2O4 (9 unsaturations) by
HREIMS. The IR absorption spectrum of 1 showed
bands characteristic of a hydroxyl (3435 cm−1), benzo-
quinone (1626 cm−1) and pyrrole (3216, 1471 cm−1)
functionalities. Two proton signals [l 12.19 (1H, s,
H-1), 5.18 (1H, d, J=5.0 Hz, 1%-OH)] were exchanged
by D2O, suggesting that 1 has an aromatic amine and a
hydroxyl group. Although a molecular composition
Keywords: Aspergillus terreus; marine algicolous fungus; terreusinone;
dipyrroloquinone; UV-A protection; quantum chemistry calculation.
* Corresponding author. Tel.: +82-51-620-6378; fax: +82-51-628-8147;
e-mail: sonbw@pknu.ac.kr
1
was analyzed to C18H22N2O4 by HREIMS, both the H
and 13C NMR spectra of 1 showed signals correspond-
ing to only 11 protons and 9 carbons for one half of the
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.08.101

7708
S. M. Lee et al. / Tetrahedron Letters 44 (2003) 7707–7710
Table 1. NMR spectral data for terreusinone (1)a,b
correlation of H-1 to C-8/4 and the downfield shift of
the carbonyl carbon signal (lC 174.0, C-4/8),7 respec-
tively. The unsymmetrical dipyrroloquinone structure
(1a) was deduced as the terreusinone from the typical
chemical shift of the carbonyl carbon (lC 174.0), rela-
tive to the symmetrical dipyrroloquinone (1b) (lC 179.3,
168.3).8
Position
lH (mult., J)
lC (mult)
HMBC
(H to C)
1
2
3
12.19 (s)
6.28 (s)
2, 3, 3a, 8, 8a
2, 3a, 4, 8a
143.4 (s)
104.4 (d)
126.0 (s)
174.0 (s)
131.3 (s)
3a
4/8
8a
1%
1%-OH
2%
To confirm the framework and dimeric orientation of 1,
the gauge-independent atomic orbital (GIAO)9 based
13C NMR chemical shifts calculations, which have
emerged as a powerful tool for structure assignments of
natural products,10 were carried out on the four pro-
posed isomers 1a–d (Fig. 1). The geometries of the four
proposed isomers were fully optimized with C2 symme-
try at the B3LYP/6-31G** level of theory. The vibra-
tional frequency analysis performed at the same level
gave no imaginary frequencies, ensuring that they are
true energy minium structures. Based on the optimized
geometries, the NMR calculations were performed at
the B3LYP/6-311G+(2d,p) level of theory. Meanwhile,
the same procedure was applied on TMS to calculate
the relative chemical shifts of these four isomers. All the
calculations were performed using the Gaussian 98
program.11
4.24 (dd, 6.0, 5.0) 71.5 (d)
5.18 (d, 5.0)
2, 3, 2%, 4%
2, 1%, 2%
2, 1%, 3%, 4%
1.91 (qqd, 6.8,
6.6, 6.0)
33.8 (d)
3%
4%
0.86 (d, 6.8)c
0.76 (d, 6.6)c
18.1 (q)d
18.7 (q)d
1%, 2%, 4%
1%, 2%, 3%
a Recorded in DMSO-d6 at 400 MHz (1H) and 100 MHz (13C).
b Assignments aided by DEPT, COSY, TOCSY, HMQC and HMBC.
c,d Interchangeable in each column.
molecule (Table 1). These data suggested that 1 is a
dimer, having a high symmetry in the molecule. The H
1
and 13C NMR data for 1, including the results from
COSY, TOCSY, DEPT, HMQC, and HMBC experi-
ments, showed the presence of a dipyrroloquinone or a
pyridone dimer, which was further supported by the
UV spectral data [355 nm (log m 3.57), 277 nm (log m
3.83)], and 1-hydroxyisobutyl groups (Table 1). The
connection of the functional groups in 1 was achieved
on the basis of HMBC correlations. Key HMBC corre-
lations from H-1% to C-2 and C-3, from H-2% to C-2,
from 1%-OH to C-2, and from H-1 to C-8/4 were critical
in establishing the one half of the plain structure of 1 as
shown.
The plots were made using experimental data versus
theoretical values, and then were analyzed with linear
regression. It was found that the correlation coefficient
(R2) for the 13C NMR spectra with the isomer 1a is
0.9963, which is higher than that for the other isomers
(0.9942, 0.9285, and 0.9557 for 1b, 1c, and 1d, respec-
tively). The computational results are summarized in
Table 2. As all the isomers have C2 symmetry struc-
tures, only a half of 13C NMR chemical shifts data have
been obtained in the calculation results. In line with the
experimental data, nine 13C NMR chemical shifts were
observed for isomers 1a, 1c and 1d, respectively. Com-
paring to isomers 1a, 1c and 1d, ten 13C NMR chemical
shifts were obtained from isomer 1b, in which the
Based on all of the foregoing evidence, four kinds of
isomers 1a–d were assumed as possible structure of 1
(Fig. 1). Among them, 4-pyridone dimer (1c and 1d)
and 2-pyridone dimer were excluded from the HMBC
Figure 1. Possible isomers of terreusinone (1).

S. M. Lee et al. / Tetrahedron Letters 44 (2003) 7707–7710
7709
Table 2. Experimentally observed 13C NMR data for 1 and values obtained by quantum chemistry calculations (ppm)a
Carbon no.b
Exptl values
Calcd values
Isomer 1b
Carbon no.c
Calcd values
Isomer 1d
Isomer 1a
Isomer 1c
2
3
3a
4/8
8a
1%
143.4
104.4
126.0
174.0
131.3
71.5
143.1
111.3
130.7
180.1
138.5
79.2
142.0
112.0
132.2
173.3, 186.1
137.2
79.1
6
5
3
4
2
149.7
131.1
124.6
166.3
177.8
79.9
147.0
135.3
146.8
167.3
158.1
80.1
7
2%
33.8
40.6
40.6
8
43.6
42.7
3%
18.1
15.2
15.2
9
13.8
13.5
4%
18.7
20.2
20.2
10
20.5
20.2
a Calculated by B3LYP/6-311+G (2d,p)//B3LYP/6-31G (d,p) method.
b Carbon number for isomers 1a and 1b.
c Carbon number for isomers 1c and 1d.
electronic environment of the C-4 was different from
C-8. So, isomer 1b was excluded. From Table 2, it can
be seen that the chemical shifts deduced by the B3LYP/
6-311+G(2d,p) program for isomer 1a are closer to the
experimental 13C NMR data, while the corresponding
values for the other isomers are more distant from the
experimental values. As a result, it became clear that
the isolated natural product has a greater probability of
adopting the structure and orientation of isomer 1a.
Based on the above evidence, the absolute stereostruc-
ture of terreusinone was determined to be 2,6-bis[(1R)-
1-hydroxyisobutyl]-1H,5H-pyrrolo[2,3-b]indole-4,8-di-
one (1). Terreusinone (1), having a dipyrroloquinone
moiety, showed a UV-A absorbing activity with ED50
value of 70 mg/mL, which is stronger than oxybenzone
currently being used as sunscreen.
Further biological evaluation of terreusinone (1) is in
progress.
The relative configuration at the C-1% position of 1 was
deduced from ROESY spectral data which showed the
correlations of H-1 to H-1%, and H-3 to 1%-OH, indica-
tive of their cis orientations, respectively.
Acknowledgements
To determine the absolute stereostructure at C-1% of 1,
we prepared benzoylated analogue from 112 and have
examined it using the CD exciton chirality method.13
The CD spectrum of benzoate derivative of 1 demon-
strated occurrence of the coupling between CD excitons
of the dipyrroloquinone and the C-1% benzoyloxy chro-
mophore.13 From the sign of the first Cotton effect at
252 nm (Dm −7.7), the C-1% configuration in 1 has been
assigned R, as depicted in Figure 2.
CD and mass spectral data were kindly provided by the
Korea Basic Science Institute. This study was sup-
ported by a grant of the Korea Health 21 R&D Project,
Ministry of Health & Welfare, Republic of Korea
(02-PJ2-PG3-21604-0003).
References
This conclusion was further supported by applying
Horeau’s method14 to 1, where the recovered a-phenyl-
butyric acid showed [h]D=+4.7° (c 0.5, benzene).15
1. Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1–47.
2. Pietra, F. Nat. Prod. Rep. 1997, 14, 453–464.
3. Li, X.; Jeong, J. H.; Lee, K. T.; Rho, J. R.; Choi, H. D.;
Kang, J. S.; Son, B. W. Arch. Pharm. Res. 2003, 26, 532.
4. The fungal strain was identified as an Aspergillus terreus
based on fatty acid methyl ester analysis (Korean Culture
Center of Microorganisms, Seoul, Korea), similarity
index 0.839.
5. The fungus was cultured for 30 days (static) at 29°C in
SWS medium: soytone (0.1%), soluble starch (1.0%), and
seawater (100%).
6. Terreusinone (1) was isolated as a yellowish solid which
showed: mp 230°C<(decomposed); [h]D=+47° (c 0.3,
MeOH); IR (KBr) 3435, 3216, 1626, 1471, 1399, 1384,
1164 cm−1; UV (MeOH) 355 nm (log m 3.57), 277 (3.83),
247 (4.08), 204 (3.73); HREIMS m/z 330.1563 [M]+ (calcd
for C18H22N2O4, 330.1580); LREIMS m/z 331 [M+H]+
(rel. int. 3), 330 [M]+ (11), 312 [M−H2O]+ (13), 294
Figure 2.

7710
S. M. Lee et al. / Tetrahedron Letters 44 (2003) 7707–7710
[M−2H2O]+ (20), 287 [M−C3H7]+ (69), 269 [M−C3H7−
Hz, H-2%), 0.97 (3H, d, J=6.7 Hz, H-3%), 0.87 (3H, d,
J=7.0 Hz, H-4%), 8.05 (2H, d, J=7.5 Hz, H-2¦), 7.55 (2H,
t, J=7.5 Hz, H-3¦), 7.68 (1H, d, J=7.5 Hz, H-4¦);
LREIMS m/z 520 [M−H2O]+ (rel. int. 2), 398 [M−H2O−
C6H5COOH]+ (2), 294 [M−(C6H5COOH×2)]+ (100), 277
(18), 146 (15), 105 (37), 91 (13).
H2O]+ (100); CD (MeOH) 374 nm (Dm −0.101), 330
(0.025), 261 (−0.252), 235 (−0.015), 219 (−0.189), 209
(0.345), 204 (−0.106); see Table 1 for NMR spectral data.
7. Shamma, M.; Hindenlang, D. M. Carbon-13 NMR Shift
Assignments of Amines and Alkaloids; Plenum Press: New
York, 1979; pp. 18–19.
8. Qiao, G. G.; Meutermans, W.; Wong, M. W.; Traubel,
M.; Wentrup, C. J. Am. Chem. Soc. 1996, 118, 3852.
9. Wolinski, K.; Hilton, J. F.; Pulay, P. J. Am. Chem. Soc.
1990, 112, 8251.
10. (a) Zhao, W.; Ye, Q.; Tan, X.; Jiang, H.; Li, X.; Chen,
K.; Kinghorn, A. D. J. Nat. Prod. 2001, 64, 1196; (b)
Helgaker, T.; Jaszunski, M.; Ruud, K. Chem. Rev. 1999,
99, 293; (c) Sebag, A. B.; Forsyth, D. A.; Plante, M. A.
J. Org. Chem. 2001, 66, 7967; (d) Betz, J.; Bauer, W. J.
Am. Chem. Soc. 2002, 124, 8699.
13. Harada, N.; Nakanishi, K. Circular Dichroic Spec-
troscopy—Exciton Coupling in Organic Stereochemistry;
Tokyo Kagaku Dojin: Tokyo, 1982; pp. 133–143.
14. Horeau, A. Tetrahedron Lett. 1961, 2, 506–512.
15. ( )-a-Phenylbutyric anhydride (15 mg) was added to a
solution of 1 (8 mg) in pyridine, and the mixture was
stirred under a nitrogen atmosphere for 48 h at rt. The
reaction mixture was partitioned into an EtOAc–aq. satd
NaHCO3 mixture. The organic phase was dried under
vacuum and the residue was chromatographed by silica
gel (n-hexane–EtOAc=5:1) to afford the ester (4.0 mg).
The aq. NaHCO3 phase was acidified with aq. 2N HCl
and extracted with EtOAc. Work-up of the EtOAc
extract in the usual manner afforded the recovered acid,
which was purified by HPLC (ODS-A, MeOH–H2O=
10:1) to furnish a-phenylbutric acid (5 mg) of [h]D=+4.7°
(c 0.5, benzene). The following data were recorded for the
ester: 1H NMR (400 MHz, CDCl3) l 9.06 (1H, br.s, H-1),
6.35 (1H, dd-like, J=2.8, 2.2 Hz, H-3), 5.48 (1H, d,
J=7.5 Hz, H-1%), 2.11 (1H, m, H-2%), 0.94 (3H, br.d,
J=6.6 Hz, H-3%), 0.85 (3H, dd, J=6.7, 1.5 Hz, H-4%), 7.26
(5H, m, Ph-a), 3.50 (1H, m, H-a), 1.60 (2H, br.s, H-b),
0.88 (3H, t, J=7.5 Hz, H-g); IR (KBr) 3193, 1736, 1635,
1463, 1261, 1078, 1019 cm−1; HREIMS m/z 623.3122
[M+H]+ (calcd for C38H43N2O6, 623.3121); LREIMS m/z
623 [M+H]+ (rel. int. 3), 622 [M]+ (11).
11. Gaussian 98, Revision A.7; Gaussian Inc.: Pitsburgh, PA,
1998.
12. A solution of 1 (10 mg) in 0.5 mL pyridine was treated
with benzoyl chloride (0.1 mL) and the whole solution
was stirred under a nitrogen atmosphere at rt for 10 min.
The mixture was poured into ice water and the whole was
extracted with EtOAc. The organic phase was washed
with satd aq. NaHCO3 and brine, and dried over MgSO4.
The product, obtained by removing the solvent under
reduced pressure, was purified by SiO2 column chro-
matography (elution with n-hexane–EtOAc 3:1) to fur-
nish the benzoate (12 mg) as a red solid. The following
data recorded the benzoate: 1H NMR (400 MHz,
DMSO-d6) l 12.76 (1H, s, H-1), 6.46 (1H, s, H-3), 5.73
(1H, d, J=7.0 Hz, H-1%), 2.32 (1H, qqd, J=7.0, 7.0, 6.7