Croissamide, a proline-rich cyclic peptide with an N-prenylated tryptophan from a marine cyanobacterium Symploca sp.

Article abstract of DOI:10.1016/j.tetlet.2018.09.016

Croissamide, a proline-rich cyclic peptide that contains an N-prenylated tryptophan, was isolated from a marine cyanobacterium Symploca sp. Its gross structure was determined by spectroscopic analyses, and the absolute configuration was established based on chiral HPLC analyses of acid hydrolysates.

Full text of DOI:10.1016/j.tetlet.2018.09.016

Tetrahedron Letters 59 (2018) 3806–3809
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Croissamide, a proline-rich cyclic peptide with an N-prenylated
tryptophan from a marine cyanobacterium Symploca sp.
Keitaro Iwasaki a, Arihiro Iwasaki a, Shimpei Sumimoto a, Takuya Sano b, Yuki Hitomi b, Osamu Ohno b,
Kiyotake Suenaga a,
⇑
a Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
b Department of Chemistry and Life Science, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Croissamide, a proline-rich cyclic peptide that contains an N-prenylated tryptophan, was isolated from a
marine cyanobacterium Symploca sp. Its gross structure was determined by spectroscopic analyses, and
the absolute configuration was established based on chiral HPLC analyses of acid hydrolysates.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 4 August 2018
Revised 30 August 2018
Accepted 6 September 2018
Available online 7 September 2018
Keywords:
Croissamide
Marine cyanobacteria
Cyclic peptide
Proline-rich
Symploca
To date, many proline-rich cyclic peptides have been discovered
from various marine organisms such as sponges, ascidians and so
on. These compounds have attracted increasing interest because
of their biological activities [1], including cytotoxicity [2], antitu-
bercular activity [3], anti-HIV activity [4], repellent (antifouling)
activity [5] and inhibitory activity toward NO production [6].
Among marine creatures, cyanobacteria are known to be prolific
producers of peptidic natural products, and several proline-rich
cyclic peptides, including wewakapeptin A [7], wewakazole B [8],
pahayokolide A [9] and trichormamide A [10], have been isolated
from marine cyanobacteria. Against this background, we investi-
gated the secondary metabolites of a marine cyanobacterium Sym-
ploca sp. and isolated croissamide (1), a cyclic peptide containing
EtOAc and H2O. The EtOAc-soluble material was further parti-
tioned between 90% aqueous MeOH and hexane. The material
obtained from the aqueous MeOH portion was subjected to frac-
tionation by reversed-phase column chromatography (ODS silica
gel, MeOH–H2O) and repeated reversed-phase HPLC to give crois-
samide (1, 10.4 mg) [11,12].
The molecular formula of 1 was found to be C67H92N12O11 by
HRESIMS (m/z 1241.7104, calcd for
C
67H93N12O11 [M+H]+
1241.7087). The NMR data for 1 are summarized in Table 1. The
1H NMR spectrum revealed the presence of a double doublet signal
(d 6.15, J = 17.7, 10.6 Hz) and two doublet signals (d 5.14,
J = 17.7 Hz, d 5.15, J = 10.6 Hz) corresponding to a mono-substi-
tuted alkene. In the 13C NMR spectrum, 11 carbonyl signals
(d 172.2, 172.1, 171.6, 170.9, 170.4, 170.1, 169.5, 169.1, 169.0,
168.8 and 166.6) were observed. Based on further analyses of the
1H NMR, 13C NMR, COSY, TOCSY, HMQC, HMBC and NOESY spectra,
11
a-amino acids, including five prolines and one N-prenylated
tryptophan. Here we report the isolation and structure determina-
tion of croissamide (1).
Marine cyanobacterial samples (1600 g, wet weight) were col-
lected at Minna Island (called ‘‘croissant island” due to its crescent
shape), Okinawa. Based on morphological observations, the
cyanobacterium was identified as Symploca sp. (see Supplementary
Data for details). This sample was extracted with methanol, and
the extract was filtered, concentrated, and partitioned between
the presence of 11 a-amino acids: glycine (Gly), alanine (Ala), two
leucines (Leu), phenylalanine (Phe), five prolines (Pro) and N-
prenylated-tryptophan (N-Pre-Trp) was confirmed. The location
of a prenyl group was determined as shown in Fig. 1 based on
two NOESY correlations: H4 of N-Pre-Trp/H15 of N-Pre-Trp and
H4 of N-Pre-Trp/H16 of N-Pre-Trp. Although significant overlap
of the methylene signals derived from the five proline residues
was observed on the 1H NMR spectrum, we were able to distin-
guish among them based on analyses of the TOCSY spectrum.
⇑
Corresponding author.
E-mail address: suenaga@chem.keio.ac.jp (K. Suenaga).
https://doi.org/10.1016/j.tetlet.2018.09.016
0040-4039/Ó 2018 Elsevier Ltd. All rights reserved.

K. Iwasaki et al. / Tetrahedron Letters 59 (2018) 3806–3809
3807
of Gly, NH of Leu1/C-1 of N-Pre-Trp, and NH of Phe/C-1 of Leu1, were
observed. Moreover, 11 NOESY correlations, H2 of Pro1/H5a of Pro2,
H2 of Pro1/H5b of Pro2, H2 of Leu2/H5a of Pro3, H2 of Leu2/H5b of
Pro3, H2 of Pro3/NH of Gly, H2 of Pro4/H5a of Pro5, H2 of Pro4/H5b
of Pro5, H2 of Pro5/NH of N-Pre-Trp, H3a of Pro5/NH of N-Pre-Trp,
H3b of Pro5/NH of N-Pre-Trp and NH of Leu1/H2 of N-Pre-Trp, were
observed. Based on these observations, the presence of two partial
sequences, Pro1 – Pro2 – Leu2 – Pro3 – Gly – Ala and Pro4 – Pro5
–
N-Pre-Trp – Leu1 – Phe, was clarified. In addition, based on the
molecular formula and the degree of unsaturation, 1 was considered
to be a cyclic peptide: Pro1 and Pro4 must be connected to Phe and
Ala, respectively. Thus, the gross structure of 1 was determined as
shown in Fig. 1.
The absolute configuration of 1 was determined as follows. The
stereochemistry of all the
a-amino acid residues was assigned to
be L-form based on chiral HPLC analyses of the hydrolysate of 1.
With regard to N-Pre-Trp, Trp was obtained due to elimination of
the prenyl group during acid hydrolysis, which was used to deter-
mine the stereochemistry.
In several solvents such as CDCl3 and CD3OD, croissamide (1)
existed as a complex mixture of several conformers, probably
due to restricted rotation of the amide bonds in the five proline
residues. However, in DMSO-d6, a single conformer of 1 was
observed. Thus, we examined the conformation of each amide
bond in the five proline residues in DMSO-d6. According to previ-
ous reports, it is possible to determine the geometries of amide
croissamide (1)
The sequences of these partial structures were determined based
on HMBC and NOESY data (Table 1 and Fig. 1). Five HMBC correla-
tions, NH of Leu2/C-1 of Pro2, NH of Gly/C-1 of Pro3, NH of Ala/C-1
Table 1
NMR data for croissamide (1) in DMSO-d6.a.
b
c
Unit
Leu1
Position
dC
dH (J in Hz)
COSY
Selected HMBC (H ? C)
Selected NOESY
1
2
3a
3b
4
5
6
NH
170.9
52.2
41.0
4.16, m
1.11, m
1.24, m
1.27, m
0.71, d (6.3)
0.80, d (6.3)
8.23, d (10.1)
3a, 3b, NH
2, 3b, 4
2, 3a, 4
3a, 3b, 5, 6
4
4
2
1
24.5
21.5
22.3
1 (N-Pre-Trp)
2 (N-Pre-Trp)
Phe
1
2
169.1d
50.6
4.70, m
2.76, dd (13.1, 7.1)
2.85, dd (13.1, 7.1)
3a, 3b, NH
2, 3b
2, 3a
1
3a
3b
4
5/9
6/8
7
38.9
4, 5, 9
4, 5, 9
137.2
129.4
128.0
126.2
7.16, m
7.23, m
7.17, m
7.32, m
6, 8
5, 9, 7
6, 8
2
NH
1 (Leu1)
1
Pro1
1
2
169.5
58.3
30.1
4.61, brd (8.4)
1.76, m
2.16, m
1.78–1.84, m
3.29, m
3.69, m
3a, 3b
5a (Pro2), 5b (Pro2)
3a
3b
4
5a
5b
2, 3b, 4
2, 3a, 4
3a, 3b, 5a, 5b
4, 5b
21.5
46.3
4, 5a
Pro2
1
2
171.6
59.1
27.6
4.40, m
1.21, m
1.87, m
1.94, m
2.10, m
3.42, m
3.53, m
3a, 3b
1
3a
3b
4a
4b
5a
5b
2, 3b, 4a, 4b
2, 3a, 4a, 4b
3a, 3b, 4b, 5a, 5b
3a, 3b, 4a, 5a, 5b
4a, 4b, 5b
25.4
47.5
2 (Pro1)
2 (Pro1)
4a, 4b, 5a
Leu2
1
2
170.1
48.0
4.68, m
3a, 3b, NH
1
5a (Pro3), 5b(Pro3)
3a
3b
4
5
6
43.1
1.20, m
1.27, m
1.50, m
0.56, d (6.2)
0.65, d (6.2)
7.81, d (9.1)
2, 3b, 4
2, 3a, 4
3a, 3b, 5, 6
4
4
2
23.5
23.3
21.8
NH
1 (Pro2)
(continued on next page)

3808
K. Iwasaki et al. / Tetrahedron Letters 59 (2018) 3806–3809
Table 1 (continued)
b
c
Unit
Pro3
Position
dC
dH (J in Hz)
COSY
Selected HMBC (H ? C)
Selected NOESY
NH (Gly)
1
2
170.4
58.9
30.3
4.45, m
1.70, m
2.12, m
1.80–1.87, m
3.44, m
3a, 3b
1
3a
3b
4
5a
5b
2, 3b, 4
2, 3a, 4
3a, 3b, 5a, 5b
4, 5b
23.9
46.6
2 (Leu2)
2 (Leu2)
3.62, m
4, 5a
Gly
Ala
1
166.6
42.1
2a
2b
NH
3.30, m
3.86, dd (18.2, 6.1)
7.56, d (6.1)
2b, NH
2a, NH
2a, 2b
1
1
1 (Pro3)
2 (Pro3)
1
2
169.0d
44.4
4.81, m
3, NH
1
3
NH
18.1
1.05, d (6.4)
8.27, d (9.6)
2
2
1 (Gly)
Pro4
1
2
168.8
58.0
30.3
4.88, brd (8.7)
2.20, m
2.00, m
1.78–1.83, m
3.33, m
3.47, m
3a, 3b
1
5a (Pro5), 5b (Pro5)
3a
3b
4
5a
5b
2, 3b, 4
2, 3a, 4
3a, 3b, 5a, 5b
4, 5b
21.5
46.6
4, 5a
Pro5
1
2
172.1
58.7
29.7
4.19, brd (7.7)
2.37, m
1.74, m
1.65, m
1.90, m
3a, 3b
NH (N-Pre-Trp)
NH (N-Pre-Trp)
NH (N-Pre-Trp)
3a
3b
4a
4b
5a
5b
2, 3b, 4a, 4b
2, 3a, 4a, 4b
3a, 3b, 4b, 5a, 5b
3a, 3b, 4a, 5a, 5b
4a, 4b, 5b
23.9
46.6
3.41, m
3.65, m
2 (Pro4)
2 (Pro4)
4a, 4b, 5a
N-Pre-Trp
1
2
3a
3b
4
5
6
7
8
172.2
51.2
29.0
4.81, m
3.09, m
3.22, m
7.23, s
3a, 3b, NH
2, 3b
2, 3a
1
NH (Leu1)
15, 16
4, 5, 6
4, 5, 6
5, 6
121.9
111.0
129.4
119.0
117.6
120.4
112.9
135.1
58.4
7.55, d (8.1)
8
5, 11
6
6.87, dd (8.1, 7.6)
6.98, dd (8.4, 7.6)
7.36, d (8.4)
7, 9
8, 10
9
9
10
11
12
13
14a
14b
15
16
NH
144.8
112.9
6.15, dd (17.7, 10.6)
5.14, d (17.7)
5.15, d (10.6)
1.61, s
1.65, s
8.17, d (10.1)
14a, 14b
13
13
12
27.6
27.4
12, 13, 16
12, 13, 15
4
4
2
2 (Pro5), 3a (Pro5), 3b (Pro5)
a
1H–13C connectivities were determined by the HMQC method.
Measured at 100 MHz.
Measured at 400 MHz.
b
c
d
These carbon signals are interchangeable.
bonds at proline residues on the basis of the 13C chemical shift
of 1, such as anti-malarial activity, protease-inhibitory activity
and anti-bacterial activity, 1 did not show any significant activities.
In conclusion, croissamide (1), a new cyclic peptide, was iso-
lated from a marine cyanobacterium, Symploca sp. The structure
of 1 was established by spectroscopic analyses and chiral HPLC
analyses of acid hydrolysates. The structure of croissamide (1)
contains five prolines and one N-prenylated tryptophan. So far, a
number of prenylated peptides have been reported, such as
hexamollamide [16], trunkamide [17] and prenylagaramides [18].
Prenylated positions of these compounds are mainly oxygen atoms
in serine, threonin and tyrosine residues. Regarding tryptophan,
C-prenylation is dominating as found in kawaguchipeptin A [19],
and there are few reports on natural compounds possessing N-
prenylated tryptophans [20]. To the best of our knowledge, 1 is
the first cyanobacterial compound that possesses an N-prenylated
difference between the proline b and
c
(
positions (
Ddb– ) [13,14].
c
The large differences observed at Pro1
D
db– = 8.6 ppm) and Pro4
c
(Ddb– = 8.8 ppm) indicated that their peptide bonds were in a cis
c
geometry as shown in Fig. 1. On the other hand, the geometries
of peptide bonds of Pro2, Pro3, and Pro5 could not be determined
clearly based on their
Ddb– values (2.2, 6.4, and 5.8 ppm for
c
Pro2, Pro3, and Pro5, respectively). However, the geometries of their
peptide bonds were determined to be trans, based on the following
NOESY correlations: H2 of Pro1 and H5a/H5b of Pro2; H2 of Leu2
and H5a/H5b of Pro3; H2 of Pro4 and H5a/H5b of Pro5.
Croissamide (1) did not inhibit the growth of human cancer
cells, HeLa and HL60, at 10 mM. Meanwhile, 1 showed weak inhibi-
tory activity against NO production in LPS-stimulated RAW 264.3
cells [15]. Although we tested for additional biological activities

K. Iwasaki et al. / Tetrahedron Letters 59 (2018) 3806–3809
3809
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[11] The detailed isolation procedures of croissamide (1) were as follows: Marine
cyanobacterial samples were collected at Minna Island, Okinawa Prefecture,
Japan, at a depth of 0–1 m in March 2018. The collected cyanobacterium
(1600 g) was extracted with methanol (3 L) for 1 week. The extract was
filtered, and the filtrate was concentrated. The residue was partitioned
between ethyl acetate (3 Â 0.3 L) and water (0.3 L). The material obtained
from the organic layer was partitioned between 90% aqueous methanol (0.3 L)
and hexane (3 Â 0.3 L). The aqueous methanol fraction (355 mg) was first
separated by column chromatography on ODS (4.0 g) eluted with 40%
methanol, 60% methanol, 80% methanol, and methanol. The fraction
(111 mg) eluted with 80% methanol was subjected to HPLC [Conditions for
HPLC separation: column, Cosmosil 5C18MS-II (/20 Â 250 mm); flow rate
5 mL/min; detection, UV 215 nm; solvent 80% MeOH] in three batches to give
a fraction that contained croissamide (1) (46.8 mg, tR = 32.0–47.6 min). This
fraction was further separated by repeated HPLC [Cosmosil 5PE-MS (/
20 Â 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 90%
Fig. 1. Gross structure of croissamide (1) based on 2D NMR correlations.
MeOH] to give croissamide (1) (10.4 mg, tR = 35.8 min).
28
[12] Croissamide (1): colorless amorphous solid; [
a
]
À108 (c 0.33, CH3OH); IR
D
(neat) 3313, 2955, 1636, 1525, 1455, 747 cmÀ1 1H NMR, 13C NMR, COSY, HMQC,
;
tryptophan. Despite several efforts, we have not yet detected any
significant biological activities of 1. Further biological evaluations
of croissamide (1) are ongoing in our laboratory.
HMBC and NOESY data, see Table 1; HRESIMS m/z 1241.7104 [M+H]+ (calcd for
C51H75N6O12, 1241.7087).
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Acknowledgement
This work was supported by JSPS KAKENHI Grant Number
18K14346.
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Appendix A. Supplementary data
[15] The inhibition % at 30 mM was 41.5 7.3%.
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Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.tetlet.2018.09.016.
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