Isolation and structural determination of the antifouling diketopiperazines from marine-derived Streptomyces praecox 291-11

Article abstract of DOI:10.1271/bbb.110943

Marine derived actinomycetes constituting 185 strains were screened for their antifouling activity against the marine seaweed, Ulva pertusa, and fouling diatom, Navicula annexa. Strain 291-11 isolated from the seaweed, Undaria pinnatifida, rhizosphere showed the highest antifouling activity and was identified as Streptomyces praecox based on a 16S rDNA sequence analysis. Strain 291-11 was therefore named S. praecox 291-11. The antifouling compounds from S. praecox 291-11 were isolated, and their structures were analyzed. The chemical constituents representing the antifouling activity were identified as (6S,3S)-6-benzyl-3-methyl-2,5-diketopiperazine (bmDKP) and (6S,3S)-6-isobutyl-3- methyl-2,5-diketopiperazine (imDKP) by interpreting the nuclear magnetic resonance and high-resolution mass spectroscopy data. Approximately 4.8mg of bmDKP and 3.1 mg of imDKP were isolated from 1.2 g of the S. praecox 291-11 crude extract. Eight different compositions of culture media were investigated for culture, the TBFeC medium being best for bmDKP and TCGC being the optimum for imDKP production. Two compounds respectively showed a 17.7 and 21 therapeutic ratio (LC₅₀/EC₅₀) to inhibit zoospores, and two compounds respectively showed a 263 and 120.2 therapeutic ratio to inhibit diatoms.

Full text of DOI:10.1271/bbb.110943

Biosci. Biotechnol. Biochem., 76 (6), 1116–1121, 2012
Isolation and Structural Determination of the Antifouling Diketopiperazines
from Marine-Derived Streptomyces praecox 291-11
y
Ji Young CHO,1; Ji Young KANG,2 Yong Ki HONG,2 Hyo Hyun BAEK,3
4
Hyoun Woong SHIN,1 and Myoung Sug KIM
1Department of Marine Biotechnology, Soon Chun Hyang University, Asan 336-745, Korea
2Department of Biotechnology, Pukyong University, Pusan 608-737, Korea
3Department of Biology, Soon Chun Hyang University, Asan 336-745, Korea
4Pathology Division, National Fisheries Research and Development Institute, Busan 619-902, Korea
Received December 9, 2011; Accepted February 29, 2012; Online Publication, June 7, 2012
[doi:10.1271/bbb.110943]
Marine derived actinomycetes constituting 185 strains
were screened for their antifouling activity against the
marine seaweed, Ulva pertusa, and fouling diatom,
Navicula annexa. Strain 291-11 isolated from the sea-
weed, Undaria pinnatifida, rhizosphere showed the high-
est antifouling activity and was identified as Streptomyces
praecox based on a 16S rDNA sequence analysis. Strain
291-11 was therefore named S. praecox 291-11. The
antifouling compounds from S. praecox 291-11 were
isolated, and their structures were analyzed. The chemi-
cal constituents representing the antifouling activity
were identified as (6S,3S)-6-benzyl-3-methyl-2,5-diketo-
piperazine (bmDKP) and (6S,3S)-6-isobutyl-3-methyl-
2,5-diketopiperazine (imDKP) by interpreting the
nuclear magnetic resonance and high-resolution mass
spectroscopy data. Approximately 4.8 mg of bmDKP and
3.1 mg of imDKP were isolated from 1.2 g of the
S. praecox 291-11 crude extract. Eight different compo-
sitions of culture media were investigated for culture, the
TBFeC medium being best for bmDKP and TCGC being
the optimum for imDKP production. Two compounds
respectively showed a 17.7 and 21 therapeutic ratio
(LC50/EC50) to inhibit zoospores, and two compounds
respectively showed a 263 and 120.2 therapeutic ratio to
inhibit diatoms.
settlement and the attachment of algal spores. In the case
of diatoms, Daume et al.6) have reported the inhibition
of a Haliotis rubra settlement by monospecific diatoma-
ceous biofilms. The unsaturated aldehydes isolated from
Thalassiosira rotula have disrupted egg development
and morphologenesis in Sphaechinus granularis, Cras-
sostrea gigas, and Calanus helgolandicus.7,8) Extracts
from Achnantes parvula and Nizchia frustulum have
respectively inhibited settling of Semibalanus bal-
anoides and the attachment of B. neritina.9) In the case
of bacteria, Pseudoalteromonas sp. biofilms have caused
significantly lower larval settlement of Bugula neritina9)
which was negatively correlated with the increasing
density of Pseudoalteromonas sp. A Pseudoalteromonas
sp. extract has inhibited cyprid settling of the barnacle,
Balamus amphitrite,10) and shown antialgal activity
toward Ulva lactuca, Chatonella sp., Gymnodium sp.,
Nitzschia paleacea, and Navicula sp.11) The ubiquinones
isolated from Alteromonas sp. KK10304 and 6-bro-
moindole-3-carbaldehyde isolated from Acinetobacter
sp. have inhibited cyprid settling of the barnacle,
Balamus amphitrite.12) Halomonas marina, Vibrio
campbelli,13) and Micrococcus sp.14) have also shown
anti-larval settlement activity against the barnacle,
Balamus amphitrite. However, only a limited number
of microorganisms have been screened for their anti-
fouling activity. Moreover, most antifouling metabolites
have not been reported from these microorganisms
and only a few have been reported as mainly unknown
high-molecular-weight carbohydrates.
Key words: antifouling; (6S,3S)-6-benzyl-3-methyl-2,5-
diketopiperazine;
(6S,3S)-6-isobutyl-3-
methyl-2,5-diketopiperazine; Streptomyces
praecox 291-11; algal spore
Marine actinomyces are a significant new chemical
resource, and many molecules with interesting bioactiv-
ities have been isolated from marine actinomycetes.15,16)
Actinomycetes possess a tremendous ability to produce
structurally diverse secondary compounds17) which often
serve as leads to develop new pharmaceuticals and
industrial chemicals. Despite the success in discovering
antibiotic and anticancer agents, marine actinomyces
have not been paid much attention for the discovery of
antifouling agents. Marine actinomycetes are obligate
marine taxa that have been found closely related to
terrestrial bacteria by a 16s rDNA sequence analysis.17)
The genus Streptomyces has been focused on due to its
ability to produce a variety of biologically active agents,
Fouling organisms cause considerable damage to the
immersed surfaces of man-made structures such as
ships, fishnets, and aquaculture facilities. Most antifoul-
ing techniques rely on a coating containing such
antifoulants as Irgarol, chlorothalonil, and diuron.1)
However, these antifoulants are toxic,2) and many
studies have reported detecting these antifoulants in
water and sediment samples in various aquatic environ-
ments.3–5)
To develop environmentally sustainable antifouling
agents, recent research has been focused on the behavior
of microorganisms, biofilm formation and metabolite
production which can inhibit marine invertebrate larval
y
To whom correspondence should be addressed. Tel: +82-41-530-1709; Fax: +82-41-530-1638; E-mail: jycho@sch.ac.kr

Diketopiperazines from Marine Streptomyces praecox 291-11
1117
Table 1. Composition of Broth Media Used for Actinomycetes Culture in 1 L Seawater (g/L) at pH 8.0
Yeast
extract
Medium
Tryptone
Casitone
Glucose
Starch
Peptone
CaCO3
Fe2(SO4)3 4H2O
KBr
Reference
TCG
3
3
3
3
5
5
5
5
4
4
4
4
Newton et al., 2008
In this study
In this study
TCGC
TBFe
TBFeC
A1
1
1
1
1
0.04
0.04
0.1
0.1
In this study
10
10
10
10
4
4
4
4
2
2
2
2
Moore et al., 1999
Choi et al., 2010
Asolkar et al., 2006
Bugni et al., 2006
A1C
A1BFe
A1BFeC
0.04
0.04
0.1
0.1
Calculation of the therapeutic ratio. The therapeutic ratio23) LC50
/
including the cytotoxic actinofuranones and azamer-
one.18,19)
EC50 was used to evaluate the antifouling efficacy of a compound in
relation to its toxicity. LC50 is the lethal dose of a compound for 50%
of the fouling organisms, whereas the EC50 is the concentration for
50% inhibition of the fouling organism. The isolated compounds were
dissolved in DMSO, and a serial two-fold dilution was made to a
concentration with no inhibitory or lethal effect. A concentration–
response curve was plotted, and a trend line was constructed for each
compound. The commercial antifouling compound, Irgarol, was
compared for its antifouling activity.
We focused this study on marine-derived Streptomy-
ces and used its metabolite to develop an antifouling
agent. The isolation and identification of the main active
antifouling compounds from the most potent S. praecox
291-11 are described, and the antifouling compounds are
evaluated. The production of these compounds from the
strain is also compared by using eight different culture
media.
Identification of the marine bacteria. Chromosomal DNA of strain
291-11 was isolated with a High Pure PCR Template Preparation kit
(Roche, Mannheim, Germany) to identify the Streptomyces strains
producing the antifouling compounds. 16S rDNA was amplified by the
polymerase chain reaction (PCR) with 27F (50-AGA GTT TGA TCC
TGG CTC AG-30) and 1492R universal primers (50-GGT TAC CTT
GTT ACG ACT T-30), and a PCR premix (Bioneer, Seoul, Korea).20)
The PCR running conditions were as follows: pre-denaturation at 94 ꢀC
for 5 min, followed by 30 cycles denaturation at 94 ꢀC for 30 s,
annealing at 55 ꢀC for 30 s, extension at 72 ꢀC for 30 s, and elongation
at 72 ꢀC for 30 s. After agarose gel electrophoresis, the PCR products
were purified with a GeneAll Gel SV kit (Generalbiosystem, Seoul,
Korea), and the purified PCR products were cloned with a TOPO-TA
cloning kit (Invitrogen, Carlsbad, CA, USA). The plasmid was isolated
with a GeneAll Plasmid SV kit (Generalbiosystem, Seoul, Korea) and
sequenced with an ABI Prism PE Big Dye Terminator Cycle DNA
sequencing kit (Applied Biosystems, Foster City, CA, USA), using an
ABI3730XL sequencer (Applied Biosystems, Carlsbad, CA, USA).
The 16S ribosomal DNA gene sequence was compared with the NCBI
gene database by using the BLASTN program.
Materials and Methods
Marine actinomycetes culture and extract. We isolated in our
previous study the marine actinomycetes from seaweed and sediment
collected from a 10-m depth along the coast of Korea (Kangnung,
Pohang, Ulsan, Pusan, Tongyoung, and Wando).20) A total of 185
strains, 87 isolated from seaweed and 98 isolated from marine
sediment, were cultured and screened for their antifouling activity. The
strains were grown in 1-L flasks containing 500 mL of a TCG
fermentation medium (Table 1) for 7 d at 25 ꢀC with 215 rpm shaking.
A 20 g/L amount of Amberlite XAD-7 resin (Fluka, Sigma, St. Louis,
MO, USA) was then added to the culture, and the slurry was shaken for
6 h. The resin was collected by filtering through cheesecloth, washed
with 1-L of deionized water to remove salts, and eluted with acetone to
yield the crude extract. Crude extracts of 85–120 mg were obtained
from the 1-L cultures, and the antifouling activity was examined. The
most active 291-11 strain was selected for additional experiments.
Inhibition assays against zoospore settlement. The fouling macro-
alga, Ulva pertusa, was collected in July 2010 from Anmyoundo, West
Sea, Korea. The spore-releasing method followed that of Cho et al.,21)
the released spores being used for the settlement assays. Slide glasses
(7:5 ꢁ 2:5 cm) were cleaned in 10% HCl for 12 h and rinsed in
deionized water for 12 h before using for U. pertusa spore settlement.
Three slide glasses were placed vertically in a 100-mL beaker, and
zoospores (7 ꢁ 106 cells) were distributed in 75 mL of seawater in the
beaker. Each bacterial extract (or compound) was dissolved in DMSO
and added to the beaker. The beakers were wrapped and placed in the
dark for 6 h at 20 ꢀC to allow the spores to settle, because U. pertusa
spores attain the stationary phase within 6 h in seawater at 20 ꢀC.21) The
number of spores that settled on each glass (4 ꢁ 2:5 cm) was counted,
and the relative rate S/C ꢁ 100 (where S is the number of settled
spores on a slide glass, and C is the number of settled spores in a
seawater reference culture) was measured.
Culture media study and scale up culture. Strain 291-11 was
cultured in eight different media to maximize the compound
production. The media compositions are shown in Table 1. The
cultures were conducted for 7 d at 25 ꢀC in 2.8-L Fernbach flasks
containing 1-L of the fermentation medium. The bacterial metabolites
were extracted from the eight different media and analyzed by liquid
chromatography-mass spectrometry (LC-MS) with a 2020 instrument
(Shimazu, Kyoto, Japan) equipped with electrospray ionization in the
positive mode. The extract was subjected to reversed-phase chroma-
tography in a Luna C8 column (4.6 mm i.d. ꢁ 10 cm, 5 mm; Phenom-
enex, Torrance, CA, USA) at a flow rate of 0.5 mL/min. The mobile
phase consisted of acetonitrile and water solvent systems. Elution was
performed with a linear gradient of 10–100% acetonitrile over 30 min.
The experiments were conducted at least three times for each
independent assay. Differences between the experimental and control
treatments were tested for significance by a one-way analysis’ of
variance (ꢀ ¼ 0:05). A scaled-up culture was conducted with strain
291-11 in 10 replicate 2.8-L Fernbach flasks containing 1-L of the
TBFeC broth medium at 25 ꢀC while shaking at 215 rpm. The cell
growth and compound production were determined every 12 h for 9 d.
Cell growth was measured by determining the number of colonies per
unit (CFU) on a solid TCG medium. The production of the target
compound was monitored by LC-MS, using the same method as that
already described. Aliquots of 20 mL of the culture medium were
withdrawn and extracted with ethyl acetate and then analyzed.
Diatom inhibition assays. The biofouling diatom, Navicula annexa,
was supplied by Korea Marine Microalgae Culture Center at Pukyong
National University, South Korea. The diatoms were cultured in an f/2
medium22) at 20 ꢀC under 70 mmol/m2/s light intensity in a 12:12
(light:dark) cycle with 50 rpm shaking. The same slide glass method as
that just mentioned was used for the inhibition assay. An initial cell
density of 2:5 ꢁ 104 cells/mL was distributed in 75 mL of the f/2
medium, and the bacterial extract was added. The number of settled
diatom cells on each glass (4 ꢁ 2:5 cm) was counted after 3 d, and the
relative rate (S/C ꢁ 100) was measured.

1118
J. Y. CHO et al.
Isolation and identification of the diketopiperazines. The crude
media to produce antifouling compounds. Bacterial
metabolites were extracted and analyzed by LC-MS,
the weight percentage of the metabolite being estimated
from the peak area on the chromatogram. The results
show that the production of compounds was signifi-
cantly higher in TCG-based cultures than that in the A1-
based culture. The concentration of bmDKP was highest
in the TBFeC medium (0.48 mg/L) and lowest in the A1
medium (0.10 mg/L). The concentration of imDKP was
highest in the TCGC medium (0.42 mg/L) and lowest in
the A1C medium (0.19 mg/L) (Fig. 1).
strain 291-11 extract was adsorbed onto diatomaceous earth (Celite)
and subjected to silica-gel flash column (100 g; 70–230 mesh)
chromatography, which was eluted with a step gradient mixture of
hexane/ethylacetate and an ethylacetate/methanol mixture to generate
9 fractions. Active fraction obtained was dissolved in methanol for
reverse-phased high performance liquid chromatography (HPLC).
Separation was achieved on a Luna C8 column (10 mm i.d. ꢁ 25 cm,
10 mm; Phenomenex). The analysis was performed on an Agilent 1200
gradient LC (Agilent Technologies, Santa Clara, CA, USA) system and
monitored at 220 nm with a ultraviolet detector. The mobile phase
consisted of acetonitrile and water solvent systems. Elution was
performed with a isocratic flow of 25% acetonitrile over 100 min at a
flow rate of 2 mL/min. Purified compounds were analyzed on a GC-
MS-QP5050A (Shimadzu) equipped with a flame ionization detector
and compared to spectral data from a database. HR-FABMS data were
obtained from a JMS HX 110 tandem mass spectrometer (Jeol, Tokyo,
Japan). The nuclear magnetic resonance (NMR) spectra were obtained
on a JNM-ECP 600 NMR spectrometer (Jeol, Tokyo, Japan), using
DMSO-d6.
Cell growth and production of the compounds
The growth curve for strain 291-11 and the antifoul-
ing compounds produced in the TBFeC medium was
measured every 12 h (Fig. 2). Strain 291-11 showed the
first phase of growth 72–132 h post inoculation. The
second phase occurred during 132–168 h, and declined
thereafter. This strain produced two target compounds
after around 84 h and production increased depending on
cell growth. The compounds produced were maximized
at the end of the second phase. bmDKP production
Absolute configuration of the amino acids. The advanced Marfey
method24,25) was applied to determine the absolute configurations of the
amino acid constituents of compounds 1 and 2. Each compound
(0.5 mg) in 6 N HCl (0.5 mL) was heated at 110 ꢀC for 16 h, and the
acid hydrolysate was evaporated to dryness. The residue was soluble in
H2O (200 mL). Half of this (100 mL) was added to 50 mL of a 1% (v/v)
solution of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) solu-
tion (Tokyo Kasei Kogyo, Tokyo, Japan) in acetone and 1 M NaHCO3
(100 mL), and then the mixture was heated to 80 ꢀC for 3 min. After
being cooled, 2 N HCl (50 mL) was added, and the solution diluted with
acetonitrile (300 mL). Each solution (10 mL) of the FDLA derivatives
was analyzed by LC-MS with positive mode electrospray ionization,
using a C8 Luna column (4.6 mm ID ꢁ 10 cm, 5 mm) at a flow rate of
0.7 mL/min with 30–50% acetonitrile in water containing 0.01 M TFA
for 50 min. Each derivative was identified by its retention time and
molecular weight when compared with FDLA standards. The retention
times (min) of the L-FDLA-converted standards were L-Ala (25.7 min),
L-Phe (31.3 min), and L-Val (26.4 min). The retention times of the
D-FDLA-converted standards were L-Ala (32.1 min), L-Phe (39.2 min),
and L-Val (40.3 min).
bmDKP
imDKP
0.5
0.4
0.3
0.2
0.1
0.0
A1
A1BFeC
TCGC
TBFe
TBFeC
A1C
A1BFe
TCG
Results
Media
Fig. 1. Antifouling Compound Production of S. Praecox 291-11 in
Eight Different Media.
The level of production was analyzed after 7 d, all experiments
being conducted five times.
Identification of the antifoulant-producing actinomycetes
Of the total of 185 strains, the crude extracts of 25
strains isolated from marine sediment and 39 strains
isolated from seaweed showed an EC50 value of less
than 20 against zoospore settlement. Strain 291-11
showing an EC50 value of 15 against zoospore settle-
ment was selected for bacterial identification by its 16S
rDNA partial sequence. Strain 291-11, which had been
isolated from the seaweed Undaria pinnatifida rhizo-
sphere, showed more than a 99% sequence similarity to
S. praecox (Table 2) based on a NCBI BLAST analysis.
This strain was therefore named S. praecox 291-11 and
was used to isolate the two antifouling diketopipera-
zines, bmDKP and imDKP.
0.5
8
7
0.4
6
0.3
0.2
0.1
0.0
5
4
3
2
1
0
Antifouling compound production in Streptomyces
Strain 291-11 was cultured in eight different media
including the TCG medium, and compound production
was measured to identify the most appropriate culture
0
24
48
72
96
120
144
168
192
216
Time (h)
Fig. 2. Cell Growth and Diketopiperazine Production by Strain 291-
11.
Cells were cultured in a TBFeC broth medium at 25 ꢀC. Cell
growth was measured on a TBFeC solid medium and is expressed as
CFU/mL ( ). bmDKP ( ) and imDKP ( ) production were
evaluated from their high-performance liquid chromatography peak
areas.
Table 2. Results of a BLAST Search Using the 16S rDNA Sequence
Strain Accession no.
Description
Max identification
99%
291-11 AB184293.1 Streptomyces praecox

Diketopiperazines from Marine Streptomyces praecox 291-11
1119
1750
1500
1250
1000
750
500
250
0
(1)
(2)
0
10
20
30
40
50
60
70
80
90
100
Time (min)
Fig. 3. High-Performance Liquid Chromatographic (HPLC) Profile for Isolating the Antifouling Compounds.
Fraction 6, collected by silica gel chromatography, was subjected to reversed-phase HPLC and eluted with an isocratic flow of 25%
acetonitrile over 100 min at a flow rate of 2 mL/min. Peaks in the HPLC profile: 1, (6S,3S)-6-benzyl-3-methyl-2,5-diketopiperazine; 2, (6S,3S)-6-
isobutyl-3-methly-2,5-diketopiperazine.
increased up to 168 h and was maximal at 0.48 mg/mL,
before immediately decreasing. imDKP production was
also maximal by 168 h with 0.31 mg/mL, but this lasted
for 12 h more, before decreasing after 180 h. Strain 291-
11 was therefore cultured in 10 replicates of 2.8-L flasks
containing 1-L of the fermentation medium and ex-
tracted after 168 h by using the method just described.
140.1 ppm. The spectrum from H-6 to H-70, amide
proton (H-1), and carbonyl carbon (C-5) revealed the
presence of a phenylalanine fragment, and the spectrum
from H-3 to H-100, amide proton (H-4), and one more
carbonyl carbon (C-2) revealed the presence of an
alanine fragment. These two fragment structures estab-
lished this compound as diketopiperazine composed of
the amino acids, phenylalanine and alanine (Fig. 4 and
Table 3). Compound 1 was identified as bmDKP from
these results. The configuration of the Ala unit was
determined by comparing the retention times of the
FDLA standards (L, 25.7 min, and D-converted,
32.1 min) with the Ala unit of the compound at
25.5 min. The respective retention times of the L- and
D-converted standards as Phe units were 31.3 and
39.2 min. The Phe unit of the compound was eluted at
31.7 min. Based on Marfey’s technique, the absolute
configuration was determined from the elution order of
the L- versus D-FDLA derivatives (Table 4). The
absolute configuration of compound 1 was therefore
L-Ala and L-Phe, and could be assigned as 3S, 6S.
Compound 1 was thus confirmed as (6S,3S)-6-benzyl-3-
methyl-diketopiperazine.
The molecular composition of antifouling compound
2 was C9H16N2O2 (positive mode, ½M þ Hꢂþ at m=z
185.11, calculated formula weight, 184.24). The 1H-
NMR spectrum revealed the presence of three methyl
protons at 1.16–1.18 (H-30, 40, 100) ppm, methylene
protons from 1.84 to 1.98 ppm (H-10), two methine
protons next to the nitrogen at 4.57 and 4.82 ppm (H-6,
H-3), one more methine proton at 1.79 ppm (H-20), and
two amide protons at 6.95 and 7.11 ppm (H-1, H-4). The
13C-NMR spectrum showed two carbonyl carbons (C-2,
C-5) at 169.8 and 168.2 ppm, three methyl carbons
(C-100, 30, 40) at 18.3–23.2 ppm, a methylene carbon
(C-10) at 41.2 ppm, two methine carbons next to the
nitrogen (C-6, C-3) at 56.1 and 52.1 ppm, and one more
methine carbon (C-20) at 23.4 ppm. The spectrum from
H-6 to H-40, amide proton (H-1), and carbonyl carbon
(C-5) revealed the presence of a valine fragment, and the
spectrum from H-3 to H-100, amide proton (H-4), and one
more carbonyl carbon (C-2) revealed the presence of an
alanine fragment. These two fragment structures estab-
lished this compound as diketopiperazine composed of
valine and alanine (Fig. 4, Table 3). These results
enabled compound 2 to be identified as imDKP. The
Isolation of the antifouling compounds
Following bioassay-guided isolation against zoospore
settlement, the extract (1.2 g) was fractionated from a
silica gel column, and active fraction 6 (97 mg) was
eluted with ethyl acetate:methanol (4:1, v/v) to show
antifouling activity with an EC50 value of 13. This
fraction was further subjected to reverse-phased HPLC.
Six peaks from HPLC were respectively generated at 8,
28, 39, 47, 65 and 78 min. The active compounds were
eluted at 39 min (compound 2) and 78 min (compound 1)
(Fig. 3). Compound 1, bmDKP, weighed 4.8 mg in a
yield of 0.4% from the 10-L culture and showed an EC50
value of 2.2. Compound 2, imDKP, weighed 3.1 mg in a
yield of 0.25% from the 10-L culture and showed an
EC50 value of 3.1. The peaks eluted at 8 and 28 min
showed very weak activity with EC50 > 300, while the
peaks eluted at 47 and 65 min showed respective EC50
values of 125 and 167.
Identification of the compounds and their antifouling
activity
The antifouling compounds isolated from S. praecox
291-11 were identified by interpreting the NMR and MS
data. The molecular composition of antifouling com-
pound 1 was C12H14N2O2 (positive mode, ½M þ Hꢂþ at
m=z 219.14, calculated formula weight of 218.25). The
1H-NMR spectrum revealed the presence of one methyl
proton at 1.67 ppm (H-100), methylene protons from 2.91
to 3.24 ppm (H-10), two methine protons next to the
nitrogen at 4.61 and 4.84 ppm (H-3, H-6), five aromatic
protons from 7.17 to 7.31 ppm (H-30–H-70), and two
amide protons at 6.95 and 7.12 ppm (H-1, H-4). The
13C-NMR spectrum showed two carbonyl carbons (C-2,
C-5) at 170.2 ppm, a methyl carbon (C-100) at 18.1 ppm,
a methylene carbon (C-10) at 38.3 ppm, two methine
carbons next to the nitrogen (C-3, C-6) at 52.4 and
55.8 ppm, five aromatic carbons (C-30–C-70) from 127.3
to 129.7 ppm, and another quaternary carbon (C-20) at

1120
J. Y. CHO et al.
Table 3. Nuclear Magnetic Resonance (NMR) Spectral Data for
Diketopiperazines
Table 5. Inhibition of Zoospore Settlement of Ulva pertusa by
Diketopiperazines
(6S,3S)-6-benzyl-3-methyl- (6S,3S)-6-isobutyl-3-methyl-
2,5-diketopiperazine
Compound (mg/mL)
LC50
EC50
LC50/EC50
Position
2,5-diketopiperazine
Compound 1
Compound 2
Irgarol
139.1
65.3
0.01
2.2
3.1
17.7
21
2
ꢁC
ꢁH
ꢁC
ꢁH
0.005
1
6.95
6.95
2
170.2
52.4
169.8
52.1
LC50 represents the lethal dose concentration for 50% of the spores.
EC50 represents the minimum inhibiting concentration for 50% of the
spores.
3
4.61
7.12
4.82
7.11
4
5
170.2
55.8
38.3
168.2
56.1
41.2
23.4
23.2
23.2
6
4.84
3.24, 2.91
4.57
Table 6. Inhibition of Diatom Settlement of Navicula annexa by
Diketopiperazines
10
20
30
40
50
60
70
100
1.84, 1.98
1.79
1.16
140.1
129.1
129.6
129.7
127.3
129.4
18.1
7.24
7.31
7.31
7.17
7.24
1.67
Compound (mg/mL)
LC50
EC50
LC50/EC50
1.16
Compound 1
Compound 2
Irgarol
210.4
132.3
0.012
0.8
1.1
263
120.2
6
0.002
18.3
1.18
LC50 represents the lethal dose concentration for 50% of the microalgae.
EC50 represents the minimum inhibiting concentration for 50% microalgae
growth.
Table 4. Retention Times (min) of D, L-FDLA Derivatized Amino
Acids of Compounds 1 and 2
though many biosynthetic classes of compounds have
been found from marine actinomycetes, only four DKPs
have been reported.28) DKPs are a relatively unexplored
class of bioactive peptides that may have great promise
in the future.29) Many recent studies have focused on
these compounds because of their significant biological
activities, including antimicrobial,30) antiviral,31) and
antifungal effects.32) Two DPKs were isolated in the
present study from the genus Streptomyces and were
active against fouling algae.
½M þ Hꢂþ
m=z
Std
FDLA
bmDKP
L-FDLA
imDKP
L-FDLA
L-Ala
L-Phe
L-Val
384.2
460.1
412.1
25.7 (L)
32.1 (D)
31.3 (L)
39.2 (D)
26.4 (L)
40.3 (D)
25.5
31.7
25.1
26.7
Biofouling in the marine environment includes pri-
mary colonization of the substrate by microorganisms
involving diatoms and macroalgal zoospores.33) Micro-
bial cells produce extracellular polymers and biofilms,34)
and marine invertebrate larvae utilize these biofilms as a
settlement indicator.35) Inhibiting this primary coloniza-
tion may be an effective way to eliminate fouling
organisms. The two DKPs were therefore assayed
against diatoms and zoospores of algae. The antifouling
activity of the two compounds in terms of their LC50
and EC50 values are shown in Tables 3 and 4. The
therapeutic ratio ðLC50=EC50Þ > 15 indicates a harmless
antifoulant.36) The two compounds inhibited diatoms
and zoospores with a >15 therapeutic ratio, suggesting
that they might be successful antifoulants, whereas
commercial compound Irgarol showed a therapeutic
ratio of <15 (Tables 3 and 4). Since both compounds
had a DKP structure, it is possible that this moiety was
an important functional group for their antifouling
activity. Both the diatom and zoospore inhibition
experiments showed bmDKP to be approximately 1.4-
fold more potent than imDKP, with EC50 values of
0.8–2.2 mg/mL, suggesting that the benzyl moiety may
have affected the bioactivity.
O
O
2
2
1''
1''
HN
HN
NH
NH
5
5
1'
1'
O
O
1
2
Fig. 4. Structures of the Diketopiperazines Isolated from the Marine
Streptomycetes praecox 291-11 Culture Extract.
1, (6S,3S)-6-benzyl-3-methyl-2,5-diketopiperazine; 2, (6S,3S)-6-
isobutyl-3-methyl-2,5-diketopiperazine.
configuration of the Ala unit was the same as that in
compound 1. The configuration of the Val unit was
determined by comparing the retention times of the
FDLA standards (L, 26.4 min, and D-converted,
40.3 min) with the Val unit of the compound at
26.7 min (Table 4). The absolute configuration of com-
pound 2 was therefore L-Ala and L-Val and was assigned
as 3S, 6S. We thus confirmed compound 2 to be (6S,3S)-
6-isobutyl-3-methyl-diketopiperazine.
bmDKP showed a therapeutic ratio for zoospore
inhibition (LC50/EC50) of 17.7, while imDKP showed
21, whereas the positive control (Irgarol) was 2
(Table 5). bmDKP showed a therapeutic ratio of 263
for diatom inhibition, imDKP showed 120.2, whereas
that of Irgarol was 6 (Table 6).
Biological metabolites isolated from marine actino-
mycetes, particularly the genus Streptomyces, have
a relatively low productivity of <1 mg/L of cul-
ture;18,19,37–40) for example, Streptomyces sp. CNB-982
produces 0.1 mg/L of anti-inflammatory cyclomarin,37)
and Streptomyces sp. YM14-060 produces approximately
0.35–0.93 mg/mL of antimicrobial piericidins.38) Wu
et al.39) have reported an amorphane productivity of
0.12–0.44 mg/mL isolated from Streptomyces sp. M491.
Discussion
Marine actinomycetes are a rich source of natural
products exhibiting such diverse biological properties as
cytotoxic, antifungal, and antibiotic activities.26,27) Al-

Diketopiperazines from Marine Streptomyces praecox 291-11
1121
However, the pigment reached 8–44 mg/mL.41) Differ-
ent medium compositions often lead to improved rates
of production of these compounds.42) Strain 291-11 in
this study produced two DKPs when using the nutri-
tional culture medium recommended for marine actino-
mycete culture to produce biological metabolites
(Table 1). The two DKPs were produced at relatively
higher levels in glucose-based cultures than those in
starch-based cultures. Adding CaCO3 to the glucose-
based culture increased imDKP, and adding two
inorganic sources (Fe2(SO4)3 4H2O and KBr) increased
the bmDKP level. Despite these observations, the cell
growth of strain 291-11 was almost the same under the
eight different culture conditions tested (data not
shown). CaCO3 added to the TCG medium (TCGC)
for the glucose-based culture resulted in an imDKP yield
of 1.44-fold that in the TCG medium. Adding Fe2(SO4)3
4H2O and KBr to the culture (TBFeC) resulted in a
bmDKP yield 1.77-fold that in the TCG medium,
whereas imDKP decreased the yield 1.35-fold compared
to that in TCGC. Adding two inorganic sources
(Fe2(SO4)3 4H2O and KBr) (A1BFe) to the starch-based
culture resulted in a slightly increased yield of the two
DKPs, whereas the other three starch-based cultures
showed a decreased DKP yield.
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