Cyclic tetrapeptides from marine bacteria associated with the seaweed Diginea sp. and the sponge Halisarca ectofibrosa

Article abstract of DOI:10.1016/j.tet.2008.01.089

A Pseudomonas sp. was cultured which was associated with the Japanese seaweed Diginea sp. Crude extracts prepared from this bacterial culture were found to inhibit the growth of other marine bacterial strains. From this bacterial culture, two new peptides cyclo-[phenylalanyl-prolyl-leucyl-prolyl] (3) and cyclo-[isoleucyl-prolyl-leucyl-alanyl] (4) have been isolated together with two known peptides (1) and (2). The crude extract from a culture of Pseudoalteromonas sp. associated with the Thai sponge Halisarca ectofibrosa was found to inhibit the growth of Bacillus subtilis and Vibrio anguillarum. Isolation studies yielded a fraction containing two peptides that were identified as cyclo-[phenylalanyl-leucyl]₂ (5) and cyclo-[leucyl-isoleucyl]₂ (6) by means of LC-MS and 2D NMR data. Absolute stereochemistry was confirmed by the synthesis of cyclo-[l-phenylalanyl-l-leucyl]₂. Peptides (1)-(3) were also isolated from this bacterial strain. None of the individual peptides isolated in this study showed antibiotic activity.

Full text of DOI:10.1016/j.tet.2008.01.089

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3147e3152
www.elsevier.com/locate/tet
Cyclic tetrapeptides from marine bacteria associated with the seaweed
Diginea sp. and the sponge Halisarca ectofibrosa
Wimolpun Rungprom a,b, Eric R.O. Siwu c, Lynette K. Lambert d, Chutiwan Dechsakulwatana e,
Michael C. Barden f, Udom Kokpol b, Joanne T. Blanchfield f, Masaki Kita g, Mary J. Garson f,
*
a Faculty of Science and Technology, Phra Nakorn Sri Ayutthaya Rajaphat University, Ayutthaya 13000, Thailand
b Natural Products Unit, Department of Chemistry, Chulalongkorn University, Bangkok 10330, Thailand
c Laboratory of Organic Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
d Centre for Magnetic Resonance, The University of Queensland, Brisbane, QLD 4072, Australia
e Institute of Marine Science, Burapha University, Chonburi 20131, Thailand
f School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, QLD 4072, Australia
g Laboratory of Bioorganic Chemistry, Department of Chemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan
Received 30 October 2007; received in revised form 9 January 2008; accepted 25 January 2008
Available online 31 January 2008
Abstract
A Pseudomonas sp. was cultured which was associated with the Japanese seaweed Diginea sp. Crude extracts prepared from this bacterial culture
were found to inhibit the growth of other marine bacterial strains. From this bacterial culture, two new peptides cyclo-[phenylalanyl-prolyl-leucyl-
prolyl](3) andcyclo-[isoleucyl-prolyl-leucyl-alanyl] (4) have been isolated togetherwithtwo knownpeptides(1) and(2). The crude extractfroma cul-
ture of Pseudoalteromonas sp. associated with the Thai sponge Halisarca ectofibrosa was found to inhibit the growth of Bacillus subtilis and Vibrio
anguillarum. Isolation studies yielded a fraction containing two peptides that were identified as cyclo-[phenylalanyl-leucyl]2 (5) and cyclo-[leucyl-
isoleucyl]2 (6) by means of LC-MS and 2D NMR data. Absolute stereochemistry was confirmed by the synthesis of cyclo-[L-phenylalanyl-L-leucyl]2.
Peptides (1)e(3) were also isolated from this bacterial strain. None of the individual peptides isolated in this study showed antibiotic activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Peptides; NMR; Sponges; Diginea; Halisarca
1. Introduction
Pro]2 1 and cyclo-[Phe-Pro]2 2 have been found in the didemnid
ascidian Cystodytes delle chiajei.4 Other examples of cyclic tet-
rapeptides have been isolated from bacteria associated with
marine sponges.5,6 In this paper we report the isolation and
stereochemistry of six cyclic tetrapeptides isolated from two
distinct bacterial strains. One bacterial strain was cultured
from the seaweed Diginea sp., while the other strain was iso-
lated from the Thai marine sponge Halisarca ectofibrosa.
Both strains were found to inhibit the growth of other marine
bacteria.
Medium ring-sized peptides containing the amino acids
leucine, isoleucine, phenylalanine, valine and proline have
been reported from diverse marine sources, including marine
microorganisms.1 The peptide cyclo-(L-Ile-L-Pro-L-Leu-L-Pro)
was isolated from an actinomycete (Nocardiopsis sp.) collected
from a Pacific deep-sea sediment.2 Crews et al. isolated fenestin
A (cyclo-(Pro-Pro-Leu-Ile)) and B (cyclo-(Pro-Val-Pro-Leu-
Ile)) from the marine sponge Leucophloeus fenestrata and sug-
gested that these metabolites might be of bacterial origin,3 while
three proline-containing cyclic peptides including cyclo-[Leu-
2. Results and discussion
A bacterial strain identified as a Pseudomonas sp. by 16S
rRNA analysis was obtained from the Japanese seaweed
* Corresponding author. Tel.: þ6133653605; fax: þ6133654273.
E-mail address: m.garson@uq.edu.au (M.J. Garson).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.089

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W. Rungprom et al. / Tetrahedron 64 (2008) 3147e3152
Diginea sp., an alga which has a symbiotic relationship with
dinoflagellates such as Amphidinium sp. The colonies inhibited
the growth of other marine bacteria when grown on seawater-
based agar medium. Purification by SiO2 column chromatogra-
phy and reversed-phase HPLC of the CHCl3eMeOH-soluble
fractions of the culture supernatant gave peptide-containing
fractions that were analyzed by 1H NMR and by LC-MS. Exten-
sive 2D NMR analysis of the individual cyclopeptide compo-
nents confirmed the isolation of the previously-characterized
peptides 1 and 24 in addition to the new peptides 3 and 4.
Peptides 1e3 were also produced by a bacterial strain, charac-
terized as a Pseudoalteromonas sp. by 16S rRNA analysis,
which was isolated from the Thai marine sponge H. ectofibrosa.
In addition the Thai bacterial culture produced the new leucine-
containing peptides 5 and 6.
five bond correlation between this upfield signal and that at dH
4.19 (Phe), and the three bond correlation between dH 4.19
and dC 170.0 ppm assigned to the Pro carbonyl, further
confirmed the placement of the Pro unit next to a Phe residue,
as did NOESY correlations between the Pro a proton and the
aromatic protons of Phe. Another correlation present in the
DQFCOSY spectrum was between dH 4.10 (Leu) and dH
4.22 (Pro). In the MS, fragment ions at m/z 245 and 211 were
consistent with ProePhe and ProeLeu fragments, respectively.
These data were consistent with cyclo-[Phe-Pro-Leu-Pro] or
with cyclo-[Phe-Leu-Pro-Pro], however, by analogy with fenes-
tin A,3 the latter structure would be expected to show a fragment
at m/z 195 for ProePro in the MS. Further, the other diprolyl
peptides isolated in this study all contained an alternate Pro-
XePro-Y sequence rather than adjacent ProePro units.
N
N
N
Ph
O O
O O
NH
Ph
O O
O O
NH
O O
O O
NH
HN
HN
Ph
HN
N
N
N
1
2
3
Ph
H
N
H
H
N
N
O O
O O
NH
O O
O O
NH
O O
O O
NH
HN
HN
HN
N
N
N
H
H
Ph
6
4
5
1
The H NMR data of 1 and 2 were recorded in CDCl3 at
600 MHz and showed significant chemical shift differences
in the a proton region to the data reported by Aracil et al.4
recorded at 360 MHz in CDCl3. Differences in 13C chemical
shift values for the a carbons were also noted. Notably, all
of the proton and carbon signals of 1 and 2 recorded at
600 MHz were observed in pairs, which suggested their C2
symmetrical conformations.
The second peptide (4) was isolated as an amorphous solid
with [a]2D3 ꢀ13.0 (c 0.05, MeOH) and showed a molecular ion
peak at m/z 417.2506 for the [MþNa] by HRESIMS consistent
with the molecular formula C20H34N4O4. In the 13C NMR
(CDCl3), in addition to signals corresponding to the side
chains of the above amino acids, four carbonyl signals at dC
170.5, 170.2, 170.1 and 167.5 ppm could be assigned as the
amide carbons of Ala, Leu, Pro and Ile, respectively, by the
3JCH correlation from their b protons. The 1H NMR, also
run in CDCl3 showed four a protons at dH 4.21 (Pro), 3.97
(Ala), 3.93 (Leu) and 3.65 (Ile) ppm, with the side chain sig-
nals for the individual amino acids assigned by DQFCOSY
data. The Ala a proton at dH 3.97 showed an HMBC correla-
tion to the amide carbonyl of Leu, while the Ile a proton at dH
3.65 showed an HMBC correlation to the amide carbonyl of
Ala. These data were fully consistent with cyclo-[Ile-Pro-
Leu-Ala].
Peptide 3 was isolated as an amorphous solid with [a]D23
ꢀ21.6 (c 0.1, MeOH) and showed a molecular ion peak at
m/z 477.2478 for the [MþNa] by HRESIMS consistent with
the molecular formula C25H34N4O4. The peptide was not sol-
uble in CDCl3 and all NMR data were therefore acquired in
CD3OD. In the 13C NMR (Table 1), in addition to signals cor-
responding to the side chains of the above amino acids, four
carbonyl signals at dC 171.5, 170.0, 167.5 and 166.0 ppm
could be assigned as the amide carbons of two Pro, one Leu
3
and one Phe, respectively, by the JCH correlation from their
By LC-MS, the HPLC fraction containing the leucine-con-
taining peptides 5 and 6 showed MHþ ions at m/z 521 and 453
for cyclo-(Phe-Leu-Phe-Leu) and cyclo-(Leu-Ile-Leu-Ile). Ions
at m/z 261 and 227 were consistent with PheeLeu and Leue
1
b protons. The H NMR (Table 1) of peptide 3 showed three
a protons at dH 4.22, 4.19 and 4.10 while a fourth a proton
was observed upfield at dH 2.59 ppm. The side chain signals
for the individual amino acids were determined by DQFCOSY
data and were fully consistent with the presence of Leu, Phe
and two different Pro residues. The Pro unit with the a proton
signal at dH 2.59 was placed adjacent to the Phe residue. The
1
Ile fragments, respectively. The H NMR (CD3OD; Table 2)
showed four a protons, those at d 4.31 and 3.65 ppm assigned
to Phe and Leu in compound 5 and those at d 3.94 and
3.84 ppm assigned to Leu and Ile in compound 6, respectively.

W. Rungprom et al. / Tetrahedron 64 (2008) 3147e3152
3149
Table 1
Proline-containing tetrapeptides isolated from marine bacteria
c-Leu-Pro-Phe-Pro 3
c-Leu-Pro-Ile-Ala 4
Residue
Residue
Leucine
Position
d
1Ha
d
13Cb
Position
d
1Hc
d
13Cd
a
b
4.10, dd, 5.6, 6.4
2.28, m
1.88, m
53.3, d
38.1, t
Leucine
a
b
3.93, dd, 4.8, 8.0
1.72, m
1.61, m
53.3
44.1
g
d (CH3)
d (CH3)
1.85, m
24.4, d
21.1
22.3
g
d (CH3)
d (CH3)
1.85, m
24.7
21.2
22.6
0.92, d, 6.4
0.92, d, 6.4
d
4.22, dd, 7.2, 8.8
2.28, m
1.99, m
0.93, d, 6.4
0.96, m, 6.4
d
4.21, dd, 7.2, 10.4
2.33, m
1.88, m
CO
a
167.5
58.9
27.7
CO
a
170.2
58.6
29.1
Proline-1
Proline
b
b
g
d
2.01, m
1.99, m
21.9
45.1
g
d
1.99, m
1.87, m
21.9
45.9
3.48, m
3.30, m
d
3.58, m
3.46, m
CO
a
171.5
58.5
39.6
CO
a
d
3.65, d, 6.4
1.84, m
170.1
62.7, d
39.9, d
Phenylalanine
4.19, dd, 4.8, 5.6
3.17, dd, 4.8, 13.6
2.95, dd, 5.6, 13.6
Isoleucine
b
b
1
135.4
g
1.57, m
1.21, m
0.99, d, 6.4
0.92, t, 7.2
d
24.9, t
2/6
3/5
4
7.20, d, 7.2
7.35, dd, 5.6, 7.2
7.35, t, 5.6
128.3
129.9
127.1
166.0
57.7
g (CH3)
d (CH3)
CO
14.8
10.7
167.2
CO
a
Proline-2
2.59, dd, 6.4, 11.2
2.00, m
1.64, m
1.89, m
1.60, m
3.48, m
3.30, m
d
Alanine
a
b
3.97, q, 7.2
1.42, d, 7.2
50.8
19.9
b
28.4
g
21.1
45.5
CO
d
170.5
d
CO
170.0
a
Measured in CD3OD relative to residual solvent at dH 3.31; 600 MHz.
Measured in CD3OD relative to residual solvent at dC 49.0; 125 MHz.
Measured in CDCl3 relative to residual solvent at dH 7.25; 600 MHz.
Measured in CDCl3 relative to residual solvent at dC 77.0; 125 MHz.
b
c
d
In CD3OH, four NH signals were apparent, those at d 8.20 and
8.14 ppm assigned to the Phe and Leu residues of 5 and those
at d 8.23 and 8.05 ppm assigned to the Leu and Ile residues of
6. The side chain signals for the four individual amino acids
were determined by the combination of DQFCOSY and
TOCSY data which were fully consistent with the presence
of Phe, Ile and two different Leu units. One of the two Leu
units showed signals at dH 0.06 and 0.87 ppm assigned to
the two diastereotopic b protons, suggesting that it could be
adjacent to a Phe residue. The second Leu showed normal
chemical shift values consistent with it being adjacent to an
Ile residue. In the 13C NMR, in addition to signals correspond-
ing to the side chains of the above amino acids, four carbonyl
signals at dC 171.8, 170.5, 169.5 and 168.8 ppm could be
assigned as the amide carbons of Leu, Leu, Ile and Phe, re-
spectively, by the 3JCH correlation from their b protons. Addi-
next to an Ile residue. In both DQFCOSY and TOCSY spectra,
five bond correlations were apparent between the a protons of
Phe and the unusual Leu residue, and between the normal Leu
and Ile, while a NOESY spectrum showed correlations from
the NH of the unusual Leu to the a proton of Phe, and from
the NH of the normal Leu to the Ile a proton. Clearly, the
two components of this fraction were cyclo-(Phe-Leu-Phe-
Leu) and cyclo-(Leu-Ile-Leu-Ile). Owing to the small sample
size, the two peptides were not further purified by reversed-
phase HPLC.
To confirm its identity, peptide 5 was prepared by solid
phase peptide synthesis using Kaiser’s 4-nitrobenzophenone
oxime resin.7 The peptide was assembled by repeated coupling
of Boc-protected amino acids in the following order: L-Phe,
L-Leu, L-Phe and finally L-Leu. Cyclization of the peptide
was achieved concomitantly with cleavage from the resin via
treatment with diisopropylethylamine and acetic acid, a pro-
cess characteristic of Kaiser resin.8 The cleavage procedure
yielded a mixture of peptide products from which cyclo-(L-
Phe-L-Leu-L-Phe-L-Leu) was purified by reversed-phase HPLC.
3
tionally, the JCH correlations between dH 4.31 and dC
170.5 ppm and between dH 3.65 and dC 168.8 ppm confirmed
the placement of Phe next to a Leu unit. Correlations between
dH 3.84 and dC 171.8 ppm and between dH 3.94 and dC
169.5 ppm confirmed the placement of the remaining Leu
1
The sample of synthetic peptide showed identical H and 13C

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W. Rungprom et al. / Tetrahedron 64 (2008) 3147e3152
Table 2
Symmetrical leucine-containing tetrapeptides isolated from Pseudoalteromonas sp.
c-Leu-Phe-Leu-Phe 5
c-Leu-Ile-Leu-Ile 6
Residue
Leucine
Position
d
1Ha
d
13Cb
d
13Cc
Residue
Leucine
Position
d
1Ha
d
13Cb
a
b
3.65, ddd, 9.9, 4.4, 1.0
0.06, m (a)
0.87, m (b)
1.42, m
53.8, d
44.9, t
54.1
45.3
a
b
3.94, ddd, 9.1, 4.4, 1.1
1.61, m (a)
1.75, m (b)
1.86, m
54.0, d
44.5, t
g
24.4, d
23.1, q
21.1, q
24.7
23.3
21.4
g
25.0, d
21.6, q
23.4, q
d (CH3)
d (CH3)
CO
0.69, d, 6.6
0.73, d, 6.6
d
d (CH3)
d (CH3)
CO
0.96, d, 6.8
0.97, d, 6.8
d
8.23, s
3.84, dd, 4.1, 1.1
1.93, m
170.5
d
170.6
d
171.8, s
d
NH
a
8.14, s
NH
a
Phenylalanine
4.31, ddd, 4.8, 3.8, 1.0
2.95, dd, 13.9, 4.8
3.28, dd, 13.9, 3.8
d
57.2, d
39.9, t
57.4
40.3
Isoleucine
60.7, d
40.3, d
b
b
1
136.6, s
136.8
g
1.25, m (a)
1.54, m (b)
1.02, d, 7.1
0.95, d, 7.6
d
2/6
3/5
4
7.20, d, 8.4
7.31, dd, 7.5, 8.4
7.26, t, 7.5
d
131.5, d
129.3, d
128.2, d
168.8, s
d
131.8
129.6
128.5
168.9
d
g (CH3)
d (CH3)
CO
15.4, q
11.9, q
169.5
d
CO
NH
NH
8.05, s
8.20, s
a
Measured in CD3OD or in CD3OH relative to residual solvent at dH 3.31; at 750 MHz.
Measured in CD3OD relative to residual solvent at dC 49.0; at 500 MHz.
Synthetic sample; at 750 MHz.
b
c
NMR data to the bacterial peptide component, including the
characteristic upfield leucine signals.
gave two new peptides cyclo-[phenylalanyl-prolyl-leucyl-
prolyl] (3) and cyclo-[isoleucyl-prolyl-leucyl-alanyl] (4)
together with two known peptides (1) and (2). Culture of
a Pseudoalteromonas sp. associated with the Thai sponge H.
ectofibrosa provided cyclo-[phenylalanyl-leucyl]2 (5) and
cyclo-[leucyl-isoleucyl]2 (6) together with (1)e(3). Despite
the crude extracts showing antibacterial activity, none of the
individually-isolated peptides were active.
The synthetic tetrapeptide 5 showed a negative optical
rotation, as do the cyclic tetrapeptides reported from marine
sources in which all the amino acid constituents are of L con-
figuration.2,5,6 In particular, the peptide (ꢀ)-cyclo-[Ile-Pro-
Leu-Pro] isolated from Nocardiopsis sp. has been shown to
contain all L amino acids.2 The peptides 3 and 4 both showed
a negative optical rotation consistent with L stereochemistry
while Marfey analysis9 of the HPLC fraction containing pep-
tides 1e3 provided L amino acid components alone. There was
insufficient of the natural peptides 5 and 6 to establish absolute
configuration of these peptides, however, on biogenetic
grounds an all L configuration is suggested for them also.
The methanol-soluble fractions of the Pseudoalteromonas
culture supernatant showed antimicrobial activity when tested
against Staphylococcus aureus, Micrococcus luteus, Bacillus
subtilis, Escherichia coli or Vibrio anguillarum. However, nei-
ther the cyclopeptide-containing fractions nor the synthetic
sample of peptide 5 showed any antibacterial activity when
screened against common bacterial targets. Other researchers
have also noted the absence of activity associated with cyclic
tetrapeptide metabolites even when the metabolites have been
isolated from extracts that show antimicrobial activity.2 This
may be ascribed to multiple ring conformations linked to
the presence of trans or cis amide bonds,10 particularly in
proline-rich cyclopeptides.11
4. Experimental
4.1. General experimental procedures
One and two dimensional NMR spectra were acquired us-
ing Jeol JNM-A600, Bruker DRX-500, Bruker DMX-750 or
JEOL JNM-ECA800 instruments. NMR spectra were obtained
in CD3OD or CD3OH at room temperature. Samples were
internally referenced to MeOH at dH 3.31 and dC 49.0. High
resolution mass measurements (accuracy within 5 ppm) were
obtained from a Finnigan MAT 900 XL-Trap electrospray
(ESI) mass spectrometer with a Finnigan API III electrospray
source. LC-MS data were obtained using either an Agilent
1100 series HPLC coupled to an PE Sciex API 3000 series
mass spectrometer with positive ion electrospray (ES) or
a PE Biosystems QSTAR mass spectrometer. Optical rotations
were recorded on a PerkineElmer 241-MC polarimeter or
a JASCO DIP-1000 polarimeter. Kaiser oxime resin, 1,3-diiso-
propylcarbodiimide (DIC) and diisopropylethylamine (DIEA)
were purchased from SigmaeAldrich Pty. Ltd. All Boc-pro-
tected amino acids, 2-benzotriazole-N,N,N0,N0-tetramethyl-
uronium-hexafluorophosphate (HBTU) and trifluroacetic acid
(TFA) were purchased from Auspep Pty. Ltd, Australia. All
solvents were distilled prior to use or were purchased of
HPLC grade.
3. Conclusions
In conclusion, the methanolic extracts of two different
marine bacterial strains have been investigated and shown to
contain cyclic tetrapeptide components. When cultured, a Pseu-
domonas sp. associated with the Japanese seaweed Diginea sp.

W. Rungprom et al. / Tetrahedron 64 (2008) 3147e3152
3151
4.2. Biological material and cultivation of bacterial strains
2.04 (6H, m), 1.92 (2H, m), 1.78 (2H, m), 1.08 (6H, d,
J¼7.0 Hz), 0.92 (6H, d, J¼7.0 Hz); 13C NMR (CDCl3,
125 MHz) d 171.9 (s, 2C), 169.0 (s, 2C), 59.2 (d, 2C), 54.1
(d, 2C), 47.1 (t, 2C), 38.2 (t, 2C), 29.6 (t, 2C), 25.1 (d, 2C),
24.2 (t, 2C), 23.0 (q, 2C), 21.9 (q, 2C).
A bacterial strain was isolated from the seaweed Diginea
sp. collected from Ishigaki Island, Okinawa, Japan together
with the dinoflagellate Amphidinium sp., by cultivation on
50% artificial seawater medium containing 0.5% proteose pep-
tone and 0.1% yeast extract (pH 7.2), and was identified by
16S rRNA gene sequence analysis as a Pseudomonas sp.
The sponge H. ectofibrosa (Demospongiae, family Halisarci-
dae) was collected near the Institute of Marine Science, Bura-
pha, in the Gulf of Thailand. The sponge is characterized by
the presence of irregular tubular chambers and the lack of
any fibrous or mineral elements in the skeleton.12 Voucher
specimens and photographs of the sponge material are avail-
able from the authors. The bacterial strain (S-9) was isolated
by cultivation on ORI medium containing 0.1% proteose pep-
tone, 0.1% yeast extract, 0.05% phytone, 0.02% sodium thio-
sulphate, 0.005% sodium sulfite and 0.005% iron(II) sulfate.
The strain was identified by 16S rRNA gene sequence analysis
as a Pseudoalteromonas sp.
4.3.2. cyclo-[Phenylalanyl-prolyl]2 (2)4
1H (CDCl3, 600 MHz) d 7.36 (4H, t, J¼7.4 Hz), 7.30 (2H, t,
J¼7.0 Hz), 7.23 (4H, d, J¼7.4 Hz), 5.64 (2H, br s), 4.28 (2H,
br d, J¼8.0 Hz), 4.09 (2H, br t, J¼8.1 Hz), 3.68e3.56 (8H,
m), 2.78 (2H, dd, J¼14.6, 10.6 Hz), 2.34 (2H, m), 2.03 (4H,
m), 1.91 (2H, m); 13C NMR (CDCl3, 125 MHz) d 169.8 (s,
2C), 165.0 (s, 2C), 139.5 (s, 2C), 129.3 (d, 2C), 129.1 (d,
2C), 127.5 (d, 2C), 59.1 (d, 2C), 56.1 (d, 2C), 45.4 (t, 2C),
36.8 (t, 2C), 28.3 (t, 2C), 22.5 (t, 2C).
4.3.3. cyclo-(Prolyl-leucyl-phenylalanyl-leucyl) (3)
1
[a]D23 ꢀ21.6 (c 0.1, MeOH); H and 13C NMR (MeOH-d4,
800 MHz), see Table 1; LC-MS 455 [MþH]þ, 245, 211; HRE-
SIMS m/z 477.2480 (calcd for C25H34NaN4O4 [MþNa]þ,
D þ0.2 mmu).
4.3. Extraction and isolation of peptides
4.3.4. cyclo-(Prolyl-leucyl-alanyl-isoleucyl) (4)
The culture broth (1.5 L) of a bacterial strain no. 27 (Pseu-
domonas sp.) was centrifuged, and the bacterial cells were
extracted with CHCl3/MeOH (1:1, 500 mL). The combined
extracts were concentrated, and the residue was partitioned
with EtOAc and water. The EtOAc layer (152 mg) showed
a potent antimicrobial activity against an orange-coloured
unidentified bacterium associated with the same host (Diginea
sp.). After further separation by Si gel chromatography and
elution using a solvent gradient of hexanes/CHCl3/EtOAc/
MeOH, the fraction eluted in EtOAc/MeOH (1:1) was sub-
jected to reverse phase HPLC using a MeOH/H2O gradient
from 50 to 100% MeOH and UV detection at 215 nm to
give the tetrapeptides 1 (1.5 mg), 2 (1.5 mg), 3 (1.0 mg) and
4 (2.0 mg).
The combined culture broth (20 L) of bacterial strain S-9
(Pseudoalteromonas sp.) was centrifuged to separate bacterial
cells from supernatant, and a portion of the supernatant (5 L)
was passed through an Amberlite XAD-2 column and eluted
with 100% MeOH, yielding an extract (60.5 mg) which dis-
played antimicrobial activity against B. subtilis and V. anguil-
larum. This extract was further fractionated on a C18 Sep Pak
eluting with MeOH/H2O 1:1 (5 mL), then with MeOH/H2O
4:1 (5 mL) and finally 100% MeOH to give three fractions
MB1 (25.5 mg), MB2 (13.5 mg) and MB3 (15.5 mg). Fraction
MB2 was further fractionated by reverse phase HPLC using
a MeOH/H2O gradient from 50 to 100% MeOH and UV detec-
tion at 220 nm to give nine fractions, including MB2-4
(1.5 mg) containing peptides 1e3 and MB2-8 (2.0 mg)
containing peptides 5 and 6.
[a]D23 ꢀ13.0 (c 0.05, MeOH); 1H and 13C NMR (MeOH-d4,
800 MHz), see Table 1; LC-MS 395 [MþH]þ; HRESIMS m/z
417.2506 (calcd for C20H34NaN4O4 [MþNa]þ, D þ2.8 mmu).
4.3.5. cyclo-[L-Phenylalanyl-L-leucyl]2 (5)
1H and 13C NMR (MeOH-d4, 750 MHz), see Table 2; LC-
MS 521 [MþH]þ, 261; HREIMS m/z 543.2954 (calcd for
C30H40NaN4O4 [MþNa]þ, D þ0.7 mmu).
4.3.6. cyclo-[L-Leucyl-L-isoleucyl]2 (6)
1H and 13C NMR (MeOH-d4, 750 MHz), see Table 2;
LC-MS 453 [MþH]þ, 227; HRESIMS m/z 475.3291 (calcd
for C24H44NaN4O4 [MþNa]þ, D þ3.1 mmu).
4.4. Marfey analysis9
Fraction MB2-4 (300 mg) containing peptides 1e3 was
hydrolyzed with 0.5 mL of 6 M HCl in a sealed ampoule
at 105 ꢁC for 12 h. The remaining HCl was removed under
a stream of N2. The resulting hydrolyzate was resuspended in
0.1% of 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (50 mL)
in acetone, then 0.1 M NaHCO3 (100 mL) was added. The reac-
tion mixture was heated at 80 ꢁC for 3 min. After cooling to
room temperature, the solution was neutralized with 0.2 M
HCl (50 mL) and diluted with MeCN/H2O/TFA(100 mL;
50:50:0.05). This solution was analyzed by reversed-phase
HPLC (AltechÔ Econosil C18; 40% MeCN in H2Oþ0.05%
TFA; 1.0 mL/min; UV detection at 340 nm). The retention
times were 5.56 and 6.06 min for authentic L- and D-proline,
18.7 and 28.6 min for L- and D-leucine and 20.0 and 32.9 min
for L- and D-phenylalanine, respectively. The retention times
for the MB2-4 hydrolyzate at 5.54, 18.7 and 20.0 min, respec-
tively, indicated the presence of L-proline, L-leucine and
L-phenylalanine.
4.3.1. cyclo-[Leucyl-prolyl]2 (1)4
1H (CDCl3, 600 MHz) d 6.61 (2H, br s, NH), 4.09 (2H, t,
J¼7.7 Hz), 3.94 (2H, br s), 3.65 (2H, dt, J¼12.1, 8.0 Hz),
3.55 (2H, td, J¼9.2, 2.9 Hz), 2.63 (2H, m), 2.38 (2H, m),

3152
W. Rungprom et al. / Tetrahedron 64 (2008) 3147e3152
4.5. Peptide synthesis
Acknowledgements
Kaiser oxime resin (1 g) with a loading capacity of 1 mmol/
g was swollen in DCM and BocePhe-OH (3.32 g, 12.5 mmol)
was attached to the resin using DIC (0.969 mL, 6.25 mmol) in
DCM as the coupling reagent and the coupling reaction
allowed to proceed overnight. After thorough washing of the
resin with DCM, MeOH and DMF, the Boc-protecting group
was removed by treatment with TFA and the resin washed
carefully and allowed to swell in DMF. The remaining amino
acids (L-Leu, L-Phe, L-Leu) were conjugated to the growing pep-
tide by stepwise coupling using standard Boc-protecting group,
solid phase peptide synthetic protocols employing HBTU/
DIEA as the activating agents and TFA/DCM for Boc-deprotec-
tion.13 When all the amino acids were assembled and the final
Boc group removed the peptidyl-resin was washed thoroughly
with DCM, i-PrOH and DMF. Cleavage and cyclization of the
peptide were achieved by treatment of the peptidyl-resin
with a solution of DIEA (0.7 mL, 4 mmol), glacial acetic acid
(0.23 mL, 4 mmol) in DMF (20 mL) for 24 h. The resin was
then removed from the solution by filtration and the resin
washed several times with DMF. The filtrate was lyophilized
to yield a crude mixture of peptides from which a sample of
the tetrapeptide (3) was purified by reverse phase HPLC using
a MeOH/H2O gradient and UV detection at 220 nm.
We thank the Australia Research Council, the Royal
Golden Jubilee Fund of Thailand, the Thailand Research
Fund, and the Japan Society for Promotion of Sciences for fi-
nancial support. We thank Dr. Sumaitt Putchakarn of the Insti-
tute of Marine Science, Burapha University for taxonomic
identification of the sponge sample, Mr. Graham MacFarlane
of the School of Molecular and Microbial Science, UQ for
the high resolution mass spectrometric measurements. We
thank Professor Daisuke Uemura, Nagoya University, for his
interest and generous support of this work.
References and notes
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1
[a]D ꢀ92.2 (c 0.57, CHCl3); H and 13C NMR (MeOH-d4,
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´
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4.6. Antibacterial assays
Assays against the bacterial strains S. aureus, M. luteus, B.
subtilis, E. coli and V. anguillarum were performed employing
the standard agar diffusion assay.
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¨
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