Indole derivatives from a marine sponge-derived yeast as DPPH radical scavengers

Article abstract of DOI:10.1021/np900483g

Two new indole derivatives (3, 4) and three known compounds (1, 2, 5) were isolated as radical scavengers from the culture filtrate of a marine sponge-derived yeast. Their structures were determined to be tyrosol (1), tryptophol (2), 2-(1H-indol-3-yl)ethy

Full text of DOI:10.1021/np900483g

J. Nat. Prod. 2009, 72, 2069–2071
2069
Indole Derivatives from a Marine Sponge-Derived Yeast as DPPH Radical Scavengers
Yasumasa Sugiyama,*,† Yuki Ito,† Motofumi Suzuki,‡ and Akira Hirota†
Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, UniVersity of Shizuoka, 52-1 Yada, Suruga-ku,
Shizuoka 422-8526, Japan, and Microbe DiVision/Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan
ReceiVed August 5, 2009
Two new indole derivatives (3, 4) and three known compounds (1, 2, 5) were isolated as radical scavengers from the
culture filtrate of a marine sponge-derived yeast. Their structures were determined to be tyrosol (1), tryptophol (2),
2-(1H-indol-3-yl)ethyl 2-hydroxypropanoate (3), 2-(1H-indol-3-yl)ethyl 5-hydroxypentanoate (4), and cyclo(L-Pro-L-
Tyr) (5) on the basis of their spectroscopic data. The absolute configurations of compounds 3 and 5 were determined
by chiral HPLC analysis combined with synthesis and Marfey’s method, respectively. Each obtained compound was
evaluated for DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, and all compounds exhibited weak
activities.
Excessive radicals are one of the risk factors for organisms,
because they damage DNA and react with other biomolecules such
as lipids and proteins. This unfavorable oxidative stress conse-
quently induces various diseases including arteriosclerosis, cardio-
vascular disease, and cancer. Therefore, antioxidants are expected
to be useful tools to combat and prevent those diseases.1,2
In our screening program for antioxidants, we have reported
several new compounds from soil-derived microorganisms3-7 and
fermented foods8,9 in the past. We continued to investigate new
antioxidants from microorganisms, and recently a yeast strain (USF-
HO25) was found as a producer of the DPPH (2,2-diphenyl-1-
picrylhydrazyl)10 radical scavenging compounds. The yeast was
separated from the marine sponge Halichondria okadai collected
at the west coast of Izu Peninsula in Shizuoka Prefecture, Japan.
The USF-HO25 strain produced several indole derivatives and
phenol derivatives in the culture filtrate. In this paper, we report
the fermentation of the USF-HO25 strain and the isolation, structural
elucidation, and radical scavenging activities of five compounds,
including two novel indole derivatives.
coupling constants in the 1H NMR spectrum of 3. The connection
of three partial structures was established by HMBC correlations
from both a methyl proton (δH 1.31 (H-3′′)) and a methylene proton
(δH 4.38 (H-2′)) to a carbonyl carbon (δC 176.5 (C-1′′)) (Figure
1). Thus, the planar structure of 3 was determined to be 2-(1H-
indol-3-yl)ethyl 2-hydroxypropanoate, a novel compound.
The absolute structure of 3 was decided by comparison of the
chromatogram of a natural product with those of synthetic
compounds. At first, we synthesized (S)-2-(1H-indol-3-yl)ethyl
2-hydroxypropanoate (3a) and (R)-2-(1H-indol-3-yl)ethyl 2-hy-
droxypropanoate (3b), which were reaction products from trypto-
phol with L-lactic acid and D-lactic acid, respectively. Subsequently,
the synthesized compounds (3a and 3b) and the natural product
(3) were analyzed by HPLC using a chiral column. Two peaks were
detected in the chromatogram of 3, and they were assigned to 3a
and 3b on the basis of their retention times. Moreover, 3b was 1.6
times larger than that of 3a. These results indicate that 3 is a mixture
of the S-form and the R-form, and the composition ratio is 5:8 (S/
R). Each of synthetic compounds 3a and 3b did not convert
spontaneously to the opposite configuration when they were purified
in a similar manner to the natural product. This phenomenon might
indicate that the mixed configuration of 3 was derived from the
biosynthetic process.
The marine sponge-derived yeast, HO-25 strain, was identified
as Pichia membranifaciens from the observation of cell and
ascospore morphology under the light microscope and the molecular
phylogenetic analysis based on the sequence of the D1/D2 region
of the 26S rRNA gene. The cultivation of the USF-HO25 strain
was conducted on a reciprocal shaker at 30 °C for 4 days. An EtOAc
extract from the culture filtrate was successively purified by silica
gel column chromatography, repeated Sephadex LH20 column
chromatography, and reversed-phase HPLC to yield compounds
1-5.
Compounds 1 and 2, respectively, were identified as tyrosol11
and tryptophol12 on the basis of their spectroscopic data. It had
been reported that both compounds were produced by a yeast, and
tyrosol showed radical scavenging activity for DPPH.13
The molecular formula of 3 was established as C13H15NO3 from
1
the HR-FABMS data. The H and 13C NMR spectra of 3 were
similar to those of 2, except that additional signals of a methyl
group (δH 1.31, δC 20.6), an oxymethine group (δH 4.22, δC 67.9),
and a carbonyl carbon (δC 176.5) were observed in the NMR spectra
of 3 (Table 1). Hence, it was supported that compound 3 was a
derivative of indole-3-ethanol. In addition, it was clear that a methyl
group is connected to an oxymethine group, as exhibited by their
The molecular formula of 4 was determined to be C15H19NO3
1
by the HR-FABMS spectrum. The H and 13C NMR spectra of 4
were comparable to those of 2, and it was suggested that 4 was an
analogue of 3. The signals of an oxymethylene moiety (δH 3.51,
δC 62.4), three methylene groups (δH 2.33, 1.63, and 1.49, δC 34.9,
22.4, and 32.9), and a carbonyl carbon (δC 175.5) were observed
besides the signals corresponding to an indole-3-ethanol moiety in
the NMR spectra of 4 (Table 1). Four methylene groups represent
an alkyl side chain, -CH2(2′′)-CH2(3′′)-CH2(4′′)-CH2(5′′)-O-,
* To whom correspondence should be addressed. Tel: +81-54-264-5555.
Fax: +81-54-264-5099. E-mail: sugiyasu@u-shizuoka-ken.ac.jp.
† University of Shizuoka.
‡ RIKEN BioResource Center.
10.1021/np900483g CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/16/2009

2070 Journal of Natural Products, 2009, Vol. 72, No. 11
Notes
Table 1. NMR Spectroscopic Data of Compounds 3 and 4 in CD3OD
3
4
δH (J in Hz)
7.08, s
b
position
δCa, mult.
δH (J in Hz)
HMBCc
3a, 7a
δC, mult.
HMBC
3a, 7a
2
3
3a
4
5
123.8, CH
111.6, qC
128.7, qC
119.2, CH
119.7, CH
122.3, CH
112.2, CH
138.1, qC
25.8, CH2
66.4, CH2
176.5, qC
67.9, CH
20.6, CH3
7.09, s
123.7, CH
111.9, qC
128.8, qC
119.3, CH
119.6, CH
122.3, CH
112.2, CH
138.1, qC
25.8, CH2
66.0, CH2
175.5, qC
34.9, CH2
22.4, CH2
32.9, CH2
62.4, CH2
7.56, d, (8.0)
7.00, t (8.0, 7.5)
7.08, td
6, 7a
3a, 7
4, 7a
3a, 5
7.55, d (8.0)
7.00, t (8.0, 7.5)
7.08, te
6, 7a
3a, 7
4, 7a
3a, 5
6
7
7.33, d (8.0)
7.32, d (8.0)
7a
1′
2′
1′′
2′′
3′′
4′′
5′′
3.10, t (6.9)
4.38, m
2, 3, 3a, 2′
3, 1′, 1′′
3.07, t (6.9)
4.32, t (6.9)
2, 3, 3a, 2′
3, 1′, 1′′
4.22, q (6.9)
1.31, d (6.9)
1′′, 3′′
1′′, 2′′
2.33, t (6.9)
1.63, quint
1.49, quint
3.51, t (6.9)
1′′, 3′′, 4′′
1′′, 2′′, 4′′, 5′′
2′′, 3′′, 5′′
3′′, 4′′
a
b
13C NMR spectrum was measured at 125 MHz. 1H NMR spectrum was measured at 500 MHz. c HMBC correlations optimized for 8 Hz are from
proton started to the indicated carbons. d Coupling constants are unclear due to overlaps. e Coupling constants are unclear due to overlaps.
Table 2. DPPH Radical Scavenging Activities of Compounds
1-5 and BHT
compounda
scavenged DPPH radical (%)c
1
2
3
4
26
52
33
47
16
86
5
Figure 1. Partial structures and HMBC correlations of compound
3.
BHTb
a Compounds were used at
a final concentration of 300 µM.
b Butylhydroxytoluene; 2,6-di-tert-butyl-4-methylphenol, used as
a
positive control. c Percentage of scavenged DPPH radical after 48 h.
of tyrosol (1) was shown in the ORAC (oxygen radical absorbance
capacity) assay, in spite of no activity for scavenging the DPPH
radical.16 High antioxidative activity of tryptophol (2) was shown
in the ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid))
assay under hydrophilic conditions.17 Many indole-containing
compounds are known to present various activities such as
anticancer, antibiotic, and anti-inflammatory activities. Accordingly,
the two novel indole derivatives (3 and 4) are expected to exhibit
other biological activities along with their antioxidative properties.
Figure 2. Partial structures and 1H-1H COSY and HMBC
correlations of compound 4.
Experimental Section
as determined by analysis of the 1H-1H COSY spectrum of 4. The
key HMBC correlations were observed from the oxymethylene
proton at δH 4.32 (H-2′) and two methylene protons (δH 2.33 (H-
2′′) and 1.63 (H-3′′)) of the alkyl chain to a carbonyl carbon (δC
175.5 (C-1′′)) (Figure 2). For this reason, it was demonstrated that
the alkyl chain was attached to an indole-3-ethanol moiety via a
carbonyl carbon (δC 175.5). Therefore, the structure of 4 was
determined to be 2-(1H-indol-3-yl)ethyl 5-hydroxypentanoate as a
novel compound.
General Experimental Procedures. Optical rotation was recorded
with a Horiba SEPA-200 spectrometer. UV spectra were recorded with
a Shimadzu UV-160A spectrometer. NMR spectra were obtained with
a JEOL JMN-ECA-500 spectrometer, using tetramethylsilane as an
internal standard. High- and low-resolution mass spectra were measured
by a JEOL JMS-700 spectrometer, using glycerol as a matrix. The
HPLC analyses were carried out on a JASCO HPLC system: PU-2080
Plus and UV-2077 Plus. HPLC for preparation was done by JASCO
880-PU and JASCO UV-970 apparatus.
Fungal Material. Morphological characteristics of the yeast strain
were examined by using standard methods described by Yarrow.18 Its
nuclear DNA was isolated from colonies on a YM agar slant incubated
at 25 °C for 4 days by using a MasterPure yeast DNA purification kit
(Epicenter Technologies, Madison, WI) according to the manufacture’s
instructions. The D1/D2 domain of the large-subunit (26S) rRNA gene
from DNA was amplified and sequenced as described by Kurtzman
and Robnett.19 A sequence-similarity search was performed by using
the BLAST 2.2.21 (Altschul et al.)20 in GenBank. The nomenclature
of the yeast strain identified follows the classification of Kurtzman.21
The D1/D2 sequence of the yeast strain was identical to that of the
type strain of Pichia membranifaciens (GenBank accession number
U75725).
HR-FABMS, H and 13C NMR, H-1H COSY, HMQC, and
HMBC spectroscopic data showed that compound 5 was a cyclic
dipeptide consisting of a proline and a tyrosine. Both chiral centers
of 5 were determined to have S configurations by HPLC analysis
of Marfey’s derivatives14 from the hydrolysate. Therefore, the
structure of 5 was identified as cyclo(L-Pro-L-Tyr).15
The five compounds (1-5) obtained in this study and BHT as a
positive control were tested for DPPH radical scavenging activities
at 300 µM (final concentration), and their percentages of scavenged
DPPH radical were calculated (Table 2). As a result, the activities
of the indolic metabolites (2, 3, and 4) were slightly higher than
the isolated phenolic compounds (1 and 5). However, between the
three indolic derivatives (2, 3, and 4) there was no significant
difference in their DPPH radical scavenging activities. Therefore,
the presence of a side chain does not affect the antioxidative activity
of an indolic derivative. In contrast, a high antioxidative activity
1
1
Fermentation. The USF-HO25 strain was inoculated into a medium
(100 mL) that consisted of glucose 1%, polypeptone 0.2%, yeast extract
0.1%, meat extract 0.1%, and NaCl 3%, pH 7.2 in a 500 mL flask; 100
flasks were inoculated and incubated at 30 °C on a reciprocal shaker
at 130 rpm for 4 days.

Notes
Isolation of DPPH Radical Scavengers from the Culture Broth
Journal of Natural Products, 2009, Vol. 72, No. 11 2071
were detected in the chromatogram of the natural product (3). The ratio
of the S/R enantiomers in the natural product mixture was calculated
from the area of their peaks.
of USF-HO25. The culture broth (10 L) was filtered, and the filtrate
was adjusted to pH 3 and extracted with EtOAc (5 L × 3). The
combined EtOAc layer was dried over anhydrous Na2SO4 and filtered,
and the solvent was removed under reduced pressure. The EtOAc extract
(1.8 g) was applied to a silica gel column and eluted with n-hexane/
EtOAc at 3:1, 1:1, and 1:3, then with EtOAc and MeOH. Two fractions
that eluted with n-hexane/EtOAc (1:1) (fraction II, 568.0 mg) and
MeOH (fraction V, 399.0 mg) showed DPPH radical scavenging activity
in the TLC assay.22 Fractions II and V were further purified three times
by Sephadex LH20 column chromatography using MeOH as an eluent.
Subfractions 17 and 18 of fraction II obtained from the third Sephadex
LH20 column were combined and further purified by preparative RP-
HPLC (column: Capcell-Pak C18 SG120, 15 × 250 mm, Shiseido;
mobile phase: 31% CH3CN/H2O), to yield compounds 1 (51.6 mg), 2
(44.3 mg), 3 (0.6 mg), and 4 (4.9 mg). Subfractions 6-11 of fraction
V were combined and evaporated to dryness in Vacuo, and the resulting
residue was purified by preparative RP-HPLC (column: Capcell-Pak
C18 SG120, 15 × 250 mm, Shiseido; mobile phase: 8% CH3CN/H2O)
to yield 5 (60.7 mg).
2-(1H-Indol-3-yl)ethyl 2-hydroxypropanoate (3): colorless oil; UV
(MeOH) λmax (log ꢀ) 222 (4.48), 282 (3.73) nm; 1H NMR, see Table 1;
13C NMR, see Table 1; HR-FABMS [M + H]+ m/z 234.1127 (calcd
for C13H16NO3, 234.1124).
2-(1H-Indol-3-yl)ethyl 5-hydroxypentanoate (4): colorless oil; UV
(MeOH) λmax (log ꢀ) 222 (4.33), 282 (3.57) nm; 1H NMR, see Table 1;
13C NMR, see Table 1; HR-FABMS [M + H]+ m/z 262.1444 (calcd
for C15H20NO3, 262.1445).
Preparations of (S)-2-(1H-Indol-3-yl)ethyl 2-hydroxypropanoate
(3a) and (R)-2-(1H-Indol-3-yl)ethyl 2-hydroxypropanoate (3b).
L-Lactic acid (270 mg) was heated in thionyl chloride (30 mL) for 6 h
at 55 °C. After the thionyl chloride was evaporated at low temperature,
CH2Cl2 (20 mL) containing tryptophol (480 mg) and 4-dimethylami-
nopyridine (366 mg) was added, and the mixture was stirred for 6 h at
room temperature. The reaction mixture was diluted with EtOAc,
washed with 0.1 M HCl, and dried over anhydrous Na2SO4. The filtrate
was evaporated to give crude materials, and they were applied to silica
gel column chromatography. The fraction was eluted with n-hexane/
EtOAc (2:1) and was further purified by preparative RP-HPLC (column:
Capcell-Pak C18 SG120, 15 × 250 mm, Shiseido; mobile phase: 32%
CH3CN/H2O) to yield 3a (47.4 mg). Similarly, (R)-2-(1H-indol-3-
yl)ethyl 2-hydroxypropanoate (3b, 4.0 mg) was prepared from D-lactic
acid (45 mg) and tryptophol (81 mg).
Measurement of DPPH Radical Scavenging Activity. DPPH
radical scavenging assay was performed by the modified method
presented previously.3-9 An ethanolic solution of the sample (750 µM,
1 mL) was mixed with a 500 µM DPPH ethanolic solution (0.5 mL)
and 0.1 M acetate buffer (pH 5.5, 1 mL). The reaction mixture was
left to stand for 48 h, at which time the absorbance of the mixture was
measured at 517 nm. The DPPH radical scavenging activity was
determined by comparison of the absorbance of sample with that of a
blank control. BHT (butylhydroxytoluene; 2,6-di-tert-butyl-4-meth-
ylphenol) was used as a positive control at the same concentration as
other samples.
Acknowledgment. We are grateful to M. Sugiyama for assistance
in collecting the marine sponge.
Supporting Information Available: NMR spectra of 1-5 and
spectroscopic data and additional information on the known compounds
are available free of charge via the Internet at http://pubs.acs.org.
References and Notes
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(S)-2-(1H-Indol-3-yl)ethyl 2-hydroxypropanoate (3a): colorless oil;
1
[R]25 -8.8 (c 0.23, MeOH); H NMR (CD3OD, 500 MHz) δ 7.56
D
(1H, d, J ) 8.0 Hz, H-4), 7.32 (1H, d, J ) 8.0 Hz, H-7), 7.09 (1H, s,
H-2), 7.08 (1H, t, H-6), 7.00 (1H, t, J ) 8.0, 7.5 Hz, H-5), 4.38 (2H,
m, H-2′), 4.22 (1H, q, J ) 6.9 Hz, H-2′′), 3.11 (2H, t, J ) 6.9 Hz,
H-1′), 1.31 (3H, d, J ) 6.9 Hz, H-3′′); 13C NMR (CD3OD, 125 MHz)
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Determination of the Configuration of Compound 3. The natural
product (3) and the synthetic compounds (3a and 3b) were analyzed
by chiral HPLC (column: Ceramospher Chiral RU-2, 4.6 × 250 mm,
Shiseido; mobile phase: MeOH; flow rate: 0.5 mL/min; detection: 280
nm). The retention times of synthetic compounds 3a and 3b were 21.9
and 17.6 min, respectively. Two peaks that correspond to 3a and 3b
NP900483G