Three new metabolites from the marine yeast Aureobasidium pullulans

Article abstract of DOI:10.1021/np980011u

Two new diketopiperazines (1 and 2) with a D-cis-4-hydroxyproline residue, and orcinotriol (3), a new 1,3-dihydroxyphenol derivative, were isolated from cultured broth of the yeast Aureobasidium pullulans, which was isolated from an Okinawan marine sponge. The structures of the compounds, including their absolute stereochemistries, were determined by spectroscopic data and chemical means.

Full text of DOI:10.1021/np980011u

696
J . Nat. Prod. 1998, 61, 696-698
Th r ee New Meta bolites fr om th e Ma r in e Yea st Au r eoba sid iu m pu llu la n s
Hideyuki Shigemori, Masami Tenma, Kengo Shimazaki, and J un’ichi Kobayashi*
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, J apan
Received J anuary 19, 1998
Two new diketopiperazines (1 and 2) with a D-cis-4-hydroxyproline residue, and orcinotriol
(3), a new 1,3-dihydroxyphenol derivative, were isolated from cultured broth of the yeast
Aureobasidium pullulans, which was isolated from an Okinawan marine sponge. The structures
of the compounds, including their absolute stereochemistries, were determined by spectroscopic
data and chemical means.
Marine microorganisms have proven to be a rich
source of compounds that might be useful for the
development of new pharmaceutical agents.1 In our
search for bioactive compounds from marine microor-
ganisms,2 two new diketopiperazines (1 and 2) and a
new 1,3-dihydroxyphenol derivative, orcinotriol (3), have
been isolated from the yeast Aureobasidium pullulans
(de Bary) Arnaud (Hyphomycetes), which was separated
from an Okinawan marine sponge. In this paper, we
describe the isolation and structure elucidation of 1-3.
The yeast A. pullulans was separated from a marine
sponge collected at Okinawa, and grown in PYG broth
[peptone (1%), yeast extract (0.5%), and glucose (2%) in
50% seawater, pH 7.0] at 25 °C for 6 days. The
supernatant of the cultured broth (12 L) was chromato-
graphed on a Diaion HP-20 column (MeOH), and the
eluent was partitioned between EtOAc and H2O. The
EtOAc-soluble portions were subjected to Si gel and
Sephadex LH-20 column chromatography to afford
compounds 1-3.
Compound 1 was obtained as a colorless amorphous
solid, and HREIMS analysis revealed the molecular
formula to be C14H16N2O3. IR absorptions of 1 implied
the presence of OH, NH, or both, (3260 cm-1) and amide
carbonyl (1650 cm-1) groups. From IR and 1H NMR
data, 1 was indicated to be a peptide. Compound 1 was
negative to the ninhydrin test, suggesting that it is a
cyclic or an N-terminus-blocked peptide. Standard
amino acid analysis of the hydrolysate of 1 (6N HCl)
gave one mole each of phenylalanine (Phe) and cis-4-
hydroxyproline (cis-Hyp). HMBC correlations of H2-9/
C-2 (δC 167.0) and NH-4/C-5 (δC 169.1) revealed that 1
was cyclo(cis-Hyp-Phe). The absolute configurations
of the cis-Hyp and Phe residues were determined to be
D- and L-, respectively, by chiral HPLC analysis (SUM-
ICHIRAL OA-5000) of the acid hydrolysate of 1. Com-
pound 1 was thus concluded to be cyclo(D-cis-Hyp-L-
Phe).
acid hydrolysate of 2 showed that 2 consisted of D-cis-
4-hydroxyproline and L-Leu. Thus, compound 2 was
determined to be cyclo(D-cis-Hyp-L-Leu).
Orcinotriol (3) was obtained as a colorless amorphous
solid and showed a molecular ion peak at m/z 168 (M+)
in the EIMS spectrum, which HREIMS analysis indi-
cated that 3 had the composition C9H12O3. IR absorp-
tions implied that 3 possesses a hydroxy group (3300
cm-1) and an aromatic ring (1600 cm-1). 1H and 13C
NMR data (δH 6.16 and 6.20; δC 102.3, 109.7, 143.3, and
160.1) indicated that 3 possesses a 1,3-dihydroxyphenol
ring. These data were similar to those of the 1,3-
dihydroxyphenol ring in R-acetylorcinol (5).4 1H-1H
COSY connectivities of H-7 to H-8 (δH 3.95) and H-8 to
H-9 suggested the presence of a 2-hydroxypropyl group.
HMBC correlations of H2-7 to C-1 indicated that the
2-hydroxypropyl group was attached at C-1. Thus, the
structure of orcinotriol was assigned to be 3. The
absolute configuration at C-8 of 3 was determined by
the modified Mosher method. Treatment of 3 with
diazomethane afforded the dimethyl ether 6, which was
then converted into the (S)- and (R)-2-methoxy-2-
trifluoromethylphenylacetic acid (MTPA) esters (7 and
8, respectively). The values of ∆δ [δ(S-MTPA ester) -
δ(R-MTPA ester)] in the 1H NMR (Figure 1) spectra
suggested that the absolute configuration at C-8 of 3
was (S).5
Compounds 1 and 2 are new diketopiperazines with
a
D-cis-4-hydroxyproline residue. Diketopiperazines
with a L-trans-hydroxyproline residue have been iso-
lated from rabbit skin tissue.6 Orcinotriol (3) is a new
1,3-dihydroxyphenol derivative related to R-acetylorci-
nol, which has been obtained from the fungus Co-
chliobolus lunata.4
Exp er im en ta l Section
Gen er a l Meth od s. Optical rotations were deter-
mined on a J ASCO DIP-370 polarimeter. UV and IR
spectra were obtained on J ASCO Ubest-35 and J ASCO
IR report-100 spectrometers, respectively. 1H and 13C
NMR spectra were recorded on Bruker ARX-500 and
AMX-600 spectrometers. The 7.26-ppm resonance of
residual CHCl3 and 77.0 ppm of CDCl3 were used as
internal references. EIMS was obtained on a J EOL DX-
303 spectrometer operating at 70 eV. FABMS was
measured on a J EOL HX-110 spectrometer by using
glycerol matrix.
The molecular formula of compound 2 was established
to be C11H18N2O3 by HREIMS. Standard amino acid
analysis of the hydrolysate of 2 gave one mole each of
leucine (Leu) and cis-Hyp. HMBC correlations of H2-
9/C-2 (δC 167.7) and NH-4/C-5 (δC 169.8) revealed that
2 was cyclo(cis-Hyp-Leu). Chiral HPLC analysis of the
* To whom correspondence should be addressed. Tel.: +81-11-706-
4985. Fax: +81-11-706-4989. E-mail: jkobay@pharm.hokudai.ac.jp.
S0163-3864(98)00011-1 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/06/1998

Notes
J ournal of Natural Products, 1998, Vol. 61, No. 5 697
) 7.2, 8.8 Hz, H-6), 2.26 (1H, m, H-7a), and 2.18 (1H,
m, H-7b); 13C NMR (CDCl3) δ 169.4 (C-5), 165.8 (C-2),
135.4 (C-11), 130.0 (C-12 and C-16), 128.8 (C-13 and
C-15), 127.6 (C-14), 67.9 (C-8), 58.8 (C-3), 55.8 (C-6), 53.7
(C-9), 40.2 (C-10), and 37.2 (C-7); EIMS m/z 260 (M+);
HREIMS m/z 260.1172 (M+) (calcd for C14H16N2O3,
260.1161).
Com pou n d 2: colorless amorphous solid; [R]24D +40.5°
1
(c 1.04, MeOH); IR (film) νmax 3280 and 1650 cm-1; H
NMR (CDCl3) δ 7.56 (1H, d, J ) 4.2 Hz, 4-NH), 4.45
(1H, m, H-8), 4.16 (1H, dd, J ) 5.8, 9.1 Hz, H-6), 3.95
(2H, m, H-3 and H-9a), 3.34 (1H, dd, J ) 4.6, 12.5 Hz,
H-9b), 2.45 (2H, m, H-7), 1.75 (1H, m, H-11), 1.63 (2H,
t, J ) 6.9 Hz, H-10), 0.98 (3H, d, J ) 6.5 Hz, H-12), and
0.95 (3H, d, J ) 6.5 Hz, H-12′); 13C NMR (CDCl3) δ 169.8
(C-5), 167.7 (C-2), 68.1 (C-8), 56.2 (C-3 or C-6), 56.1 (C-6
or C-3), 54.0 (C-9), 42.2 (C-10), 36.8 (C-7), 24.6 (C-11),
23.0 (C-12), and 21.5 (C-12′); EIMS m/z 226 (M+);
HREIMS m/z 226.1290 (M+) (calcd for C11H18N2O3,
226.1318).
F igu r e 1. 1H NMR chemical shift differences (∆δ) for MTPA
esters of compound 6; ∆δ (ppm) ) δ[(S)-MTPA ester (7)] -
δ[(R)-MTPA ester (8)].
Or cin otr iol (3): colorless amorphous solid; [R]25
D
+6.0° (c 1.1, MeOH); UV (MeOH) λmax 276 (ꢀ 5900) and
208 (17 000) nm; IR (film) νmax 3300 and 1600 cm-1; 1H
NMR (CD3OD) δ 6.20 (2H, d, J ) 2.1 Hz, H-2 and H-6),
6.16 (1H, t, J ) 2.1 Hz, H-4), 3.95 (1H, m, H-8), 2.70
(1H, dd, J ) 6.4, 13.2 Hz, H-7a), 2.50 (1H, dd, J ) 6.8,
13.2 Hz, H-7b), and 1.17 (3H, d, J ) 6.2 Hz, H-9); 13C
NMR (CD3OD) δ 160.1 (C-3 and C-5), 143.3 (C-1), 109.7
(C-2 and C-6), 102.3 (C-4), 70.6 (C-8), 47.7 (C-7), and
23.7 (C-9); EIMS m/z 168 (M+), 153, 123, and 110;
HREIMS m/z 168.0775 (M+) (calcd for C9H12O3,
168.0787).
Am in o Acid An a lysis of Hyd r olysa te of 1. Com-
pound 1 or 2 (0.05 mg, each) was dissolved in 6N HCl
(100 mL) and heated to 110 °C for 24 h in a sealed tube.
Of cis-Hyp and Phe 1 mol each was found in the
hydrolysate of 1, while 1 mol each of cis-Hyp and Leu
was found in the hydrolysate of 2 by standard amino
acid analysis.
Collection a n d Cu ltiva tion . The yeast A. pullulans
was separated from an unidentified marine sponge
collected in Okinawa. Subcultures of the organism are
deposited at Faculty of Pharmaceutical Sciences, Hok-
kaido University. The yeast was grown in the PYG
broth [peptone (1%), yeast extract (0.5%), and glucose
(2%) in 50% seawater] at 25 °C for 6 days. The cultured
broth (12 L) was centrifuged at 5000 rpm for 20 min.
Extr a ction a n d Sep a r a tion . The aqueous super-
natant of the cultured broth (12 L) was passed through
a Diaion HP-20 column (Mitsubishi Chemicals, Co.,
Ltd.), which was subsequently eluted with MeOH. The
eluent was partitioned between EtOAc and H2O. The
EtOAc-soluble portions were subjected to a Si gel
column (CHCl3-MeOH, 90:10) to afford two fractions,
a (49 mg) and b (43 mg). Fraction b was separated by
a Si gel column (CHCl3-Me2CO) to give compounds 1
(10.2 mg) and 2 (9.7 mg), and orcinotriol (3, 8.2 mg).
Fraction a was subjected to a Si gel column (CHCl3-
EtOAc) to afford orcinol (4, 1.2 mg) and R-acetylorcinol
(5, 2.2 mg).
Deter m in a tion of Absolu te Con figu r a tion of 1
a n d 2. Compound 1 or 2 (0.1 mg, each) was hydrolyzed
with 6N HCl aqueous at 110 °C for 18 h in a sealed tube.
The residue was dissolved in H2O for chiral HPLC
analysis. The chiral HPLC analysis was carried out
using a SUMICHIRAL OA-5000 column [Sumitomo
Chemical Industry, 4 × 150 mm; flow rate, 0.5 mL/min;
eluent, MeOH-H2O (15:85) containing 2.0 mmol CuSO4;
detection, UV at 254 nm; column temperature, 40 °C].
Retention times of standard L- and D-cis-Hyp, L- and
D-Phe, and L- and D-Leu were 5.2, 3.6, 22.1, 30.0, 9.9,
and 13.7 min, respectively, and those of hydrolysis
products of 1 were found to be 3.6 and 22.1 min, and
those of hydrolysis products of 2 were 3.6 and 9.9 min.
Dim eth yl Eth er (6) of Or cin otr iol (3). To a MeOH
solution (100 µL) of compound 3 (1.1 mg), CH2N2-Et2O
solution (200 µL) was added at room temperature for 7
h. After evaporation of solvent, the residue was passed
through a Si gel column (hexane-EtOAc, 1:1) to afford
the dimethyl ether (6, 0.7 mg); 1H NMR (CD3OD) δ 6.42
(2H, d, J ) 2.2 Hz, H-2 and H-6), 6.36 (1H, t, J ) 2.2
Hz, H-4), 3.99 (1H, m, H-8), 3.79 (6H, s, MeO × 2), 2.76
(1H, dd, J ) 6.7, 13.3 Hz, H-7a), 2.62 (1H, dd, J ) 6.4,
13.3 Hz, H-7b), and 1.18 (3H, d, J ) 6.2 Hz, H-9); EIMS
m/z 196 (M+).
Com pou n d 1: colorless amorphous solid; [R]24D +34.7°
(c 1.02, MeOH); UV (MeOH) λmax 281 (ꢀ 2900) and 208
(28 000) nm; IR (film) νmax 3260 and 1650 cm-1; 1H NMR
(CDCl3) δ 7.1-7.3 (5H, m, H-12-H-16), 6.82 (1H, d, J
) 3.3 Hz, 4-NH), 4.31 (1H, m, H-8), 4.22 (1H, m, H-3),
3.87 (1H, d, J ) 12.6 Hz, H-9a), 3.21 (1H, dd, J ) 4.9,
12.6 Hz, H-9b), 3.14 (1H, dd, J ) 6.3, 13.7 Hz, H-10a),
3.04 (1H, dd, J ) 4.3, 13.7 Hz, H-10b), 2.95 (1H, dd, J

698 J ournal of Natural Products, 1998, Vol. 61, No. 5
Notes
(S)-MTP A Ester (7) of Com p ou n d 6. To a CH2Cl2
solution (100 µL) of compound 6 (0.3 mg), DMAP (1.0
mg), and Et3N (2 µL) was added (R)-(-)-MTPACl (2.0
mg) at room temperature, and stirring was continued
for 3 h. After evaporation of solvent, the residue was
purified by Si gel TLC (hexane-EtOAc, 2:1) to afford
the (S)-MTPA ester (7) as colorless oil: 1H NMR (CDCl3)
δ 7.3-7.5 (5H, m, Ph), 6.364 (2H, d, J ) 2.2 Hz, H-2
and H-6), 6.358 (1H, t, J ) 2.2 Hz, H-4), 5.393 (1H, m,
H-8), 3.732 (6H, s, MeO × 2), 3.413 (3H, s, MeO of
MTPA), 2.915 (1H, dd, J ) 8.4, 13.9 Hz, H-7a), 2.831
(1H, dd, J ) 5.4, 13.9 Hz, H-7b), and 1.332 (3H, d, J )
6.2 Hz, H-9); EIMS m/z 412 (M+).
(R)-MTP A Ester (8) of Com p ou n d 6. Compound
6 (0.3 mg) was treated with (S)-(+)-MTPACl (2.0 mg)
by the same procedure as described above to afford the
(R)-MTPA ester (8) as colorless oil: 1H NMR (CDCl3) δ
7.3-7.5 (5H, m, Ph), 6.312 (1H, t, J ) 2.2 Hz, H-4), 6.288
(1H, 2H, d, J ) 2.2 Hz, H-2 and H-6), 5.414 (1H, m,
H-8), 3.716 (6H, s, MeO × 2), 3.481 (3H, s, MeO), 2.892
(1H, dd, J ) 7.2, 13.8 Hz, H-7a), 2.747 (1H, dd, J ) 6.0,
13.8 Hz, H-7b), and 1.355 (3H, d, J ) 6.3 Hz, H-9); EIMS
m/z 412 (M+).
Ack n ow led gm en t. The authors thank Dr. Y. Mi-
kami (Chiba University) for identification of the yeast.
This work was partly supported by a grant-in-aid from
the Naito Foundation and a grant-in-aid for scientific
research from the Ministry of Education, Science,
Sports, and Culture of J apan.
Refer en ces a n d Notes
(1) Fenical, W. Chem. Rev. 1993, 93, 1673-1683, and references
therein.
(2) Kobayashi, J .; Mikami, S.; Shigemori, H.; Takao, T.; Shimonishi,
Y.; Izuta, S.; Yoshida, S. Tetrahedron 1995, 51, 10487-10490.
(3) Pettersson, G. Acta Chem. Scand. 1964, 18, 1202-1207.
(4) Nukita, M.; Marumo, S. Agric. Biol. Chem. 1977, 41, 717.
(5) Ohatani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am.
Chem. Soc. 1991, 113, 4092-4095.
(6) Ienaga, K.; Nakamura, K.; Goto, T. Tetrahedron Lett. 1987, 28,
1285-1286.
NP980011U