Hapalocyclamide: A novel phytotoxic hexapeptide of the cyanobacterium Hapalosiphon sp.

Article abstract of DOI:10.1016/j.tetlet.2011.12.048

Hapalocyclamide, a novel oxazole-, thiazole- and thiazoline-containing cyclic hexapeptide, was isolated from the terrestrial cyanobacterium Hapalosiphon sp., and which showed phytotoxic activity on lettuce seedling growth. The gross structure of hapalocyclamide was established from spectroscopic data and chemical degradation. The absolute stereochemistry was determined by Marfey's analysis. Hapalocyclamide was established as cyclo-thiazole-l-alanine-oxazole-d-alanine-d-thiazoline-d-phenylalanine.

Full text of DOI:10.1016/j.tetlet.2011.12.048

Tetrahedron Letters 53 (2012) 977–979
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Hapalocyclamide: a novel phytotoxic hexapeptide of the cyanobacterium
Hapalosiphon sp.
⇑
Intira Koodkaew, Shigeru Matsuyama , Yukari Sunohara, Hiroshi Matsumoto
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hapalocyclamide, a novel oxazole-, thiazole- and thiazoline-containing cyclic hexapeptide, was isolated
from the terrestrial cyanobacterium Hapalosiphon sp., and which showed phytotoxic activity on lettuce
seedling growth. The gross structure of hapalocyclamide was established from spectroscopic data and
chemical degradation. The absolute stereochemistry was determined by Marfey’s analysis. Hapalocycla-
Received 16 November 2011
Revised 9 December 2011
Accepted 12 December 2011
Available online 19 December 2011
mide was established as cyclo-thiazole-
L
-alanine-oxazole-
D
-alanine-
D-thiazoline-D-phenylalanine.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Hapalosiphon sp.
Cyclic peptide
Hapalocyclamide
Phytotoxicity
Cyclic peptides are a class of cyanobacterial metabolites that
incorporate peptides or possess peptide-substructures containing
a range of proteinogenic and nonproteinogenic amino acids with
a high level of structural variation.1,2 Cyanobactin was proposed
as a collective name for cyclic peptides that contain heterocyclized
amino acids or isoprenoid amino acid derivatives. Cyanobactins
were initially defined as containing oxazolines, thiazolines, or their
oxidized derivatives oxazoles and thiazoles. To date, more than 100
cyanobactins have been identified from symbiotic associations
between cyanobacteria and ascidians or from free-living cyanobac-
teria.3 As part of our continuing effort to isolate phytotoxic com-
pounds from cultured cyanobacteria, we report here the isolation
and structural elucidation of a novel cyclic hexapeptide, hapalocy-
clamide4 (1) (Fig. 1), from the terrestrial cyanobacterium Hapalosi-
phon sp., Stigonemataceae.
molecule. High resolution mass spectra of these ions demonstrated
the molecular formulas as C25H26N6O4S2 (observed 538.1471; calcd
538.1457) and C18H19N6O4S2 (observed 447.0905; calcd 447.0909).
Intense absorptions in the IR spectrum at 3398 cmꢀ1 (NH
stretching vibration of secondary amide), 1675 cmꢀ1 (amide-I
band), and 1520 cmꢀ1 (amide-II band) and its lipophilic nature
suggested that 1 is a cyclic peptide. In addition, the 1H NMR spec-
trum showed the presence of three broad doublets at d 7.45, 8.39,
and 8.66 ppm (Table 1) that were attributed to amide NH signals of
amino acid, indicating that the compound was a peptide.
The products of acid hydrolysis,6 which were converted into
methyl esters and N-acetylated for GC–MS analysis, afforded phen-
ylalanine, alanine, cysteine, and thiazole-containing amino acids (2,
3) (Fig. 2). The first three amino acids were identified by comparison
The Hapalosiphon sp., isolated from soil collected in Bangkok,
Thailand, was cultured in controlled conditions.5 Dried cells
(20 g) were extracted with MeOH, and the resulting extract was
fractionated using a silica gel column, an ODS column and prepara-
tive HPLC, guided by a phytotoxicity bioassay. The active fraction
was applied to silica gel column and eluted with hexane–EtOAc
(1:1) and EtOAc to afford hapalocyclamide (1) (44.0 mg, 0.220%
yield based on dry cyanobacterial mass).
Hapalocyclamide (1) was isolated as a colorless needle-like crys-
tal. Direct inlet electron impact mass spectrum showed the molec-
ular ion at m/z 538 (M+, 86%) and a characteristic fragment ion at m/
z 447 (100%, M+-91) indicating the loss of a benzyl group from the
⇑
Corresponding author. Tel.: +81 29 853 4686; fax: +81 29 853 4605.
E-mail address: honeybee@sakura.cc.tsukuba.ac.jp (S. Matsuyama).
Figure 1. Structure of hapalocyclamide (1).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.048

978
I. Koodkaew et al. / Tetrahedron Letters 53 (2012) 977–979
Table 1
The nitrogen-bearing methine [d 5.30 (H-18), 52.5 (C-18)] had cor-
1H and 13C NMR data for hapalocyclamide (1)a
relations with an amide proton [d 8.39 (NH-3)] and methylene [d
3.28, 3.44 (2H-19), 39.1 (C-19)]. The methylene protons were cor-
related through an HMBC experiment to a phenyl ring system [d
7.10 (H-21, 24), 128.1 (C-21, 24); d 7.11 (H-22, 25), 129.7 (C-22,
25); d7.05 (H-23), 126.9 (C-23)].
No.
dC
dH Mb, Jc
HMBC
1
2
3
4
5
6
159.3 qC
148.0 qC
124.7 CH
171.7 qC
47.7 CH
C-3, NH-3
C-3
NH-3
C-3, 5, 6, NH-1
C-3, 6, NH-1
C-5
8.23 s
The singlet aromatic proton [d 8.23 (H-3), 124.7 (C-3)] exhibited
a signal for thiazole. The thiazoline ABX spin system was estab-
lished by COSY correlations of nitrogen-bearing methine [d 4.91
(H-15), 77.8 (C-15)] with sulfur-bearing methylene [d 3.38, 3.83
(2H-16), 36.8 (C-16)]. HMBC experiments demonstrated the corre-
lation of C-16 with C-15 and C-17 to confirm the thiazoline system.
The last modified amino acid was identified as methyloxazole. The
oxygen-bearing carbon [d 128.5 (C-9)] had a correlation with a
methyl group [d 2.67 (3H-10), 11.6 (C-10)] and a quaternary carbon
[d 153.8 (C-8)]in HMBC findings. In addition, the chemical shifts of
the thiazole amino acid unit (Ala-Tzl) and methyloxazole amino
acid unit (Ala-mOzl) were in good agreement with those reported
for dendroamide8 and venturamide.11
5.41 qui, 6.61
1.70 d, 6.70
8.66 d, 6.15
24.3 CH3
NH-1
7
8
160.6 qC
153.8 qC
128.5 qC
11.6 CH3
161.0 qC
44.0 CH
C-5, 10, NH-1
C-10
C-10
9
10
11
12
13
NH-2
14
15
16
2.67 s
C-12, 13, NH-2
C-13, NH-1
C-12
4.97 qui, 6.83
1.45 d, 6.80
7.45 d, 6.85
19.9 CH3
169.7 qC
77.8 CH
36.8 CH2
C-15, 16, NH-2
C-16, NH-2
C-15
4.91 ddd, 12.14, 9.46, 2.46
3.38 dd, 12.28, 11.28
3.83 dd, 11.20, 9.25
17
18
19
172.5 qC
52.5 CH
39.1 CH2
C-16, 18, 19, NH-3
C-19, NH-3
C-21
HMBC data (Table 1 and Fig. 3) provided information on the
amino acid sequence, thus constructing the cyclic hexapeptide
structure of hapalocyclamide (1). Correlation from C-7 to H-10
established the linkage of the first alanyl residue of 3 through
the oxazole ring. C-11 of oxazole exhibited correlations with H-
12, H-13 and NH-2, thus establishing the connection of the oxazole
ring to the second alanyl residue. The second alanyl residue was
linked to the thiazoline ring through HMBC correlation of C-14
with H-15 and H-16. Finally, the correlation of C-17 with H-18,
H-19 and NH-3 established the connection of thiazoline through
the benzyl residue of 3 to close the macrocyclic ring.
5.30 m
3.28 dd, 13.93, 3.68
3.44 dd, 13.93, 4.85
20
135.3 qC
128.1 CH
129.7 CH
126.9 CH
C-18, 19, 21, 22
C-19, 22, 23
C-21, 23
21, 24
22, 25
23
7.10 m
7.11m
7.05m
8.39 d 7.55
C-21, 22
NH-3
Recorded in CDCl3 at 150 MHz for 13C and 600 MHz for 1H using TMS as internal
standard.
a
b
M = Multiplicity.
J in Hz.
c
The absolute configurations of the chiral centers of amino acids
in 1 were analyzed by the Marfey’s method9,10 after hydrolysis (1 h
and 19 h). This procedure established the presence of
from the thiazoline ring. Only the oxazole ring was cleaved by
direct hydrolysis to generate -alanine. After hydrolysis for 1 h,
-phenylalanine was detected; however, racemization of phenylal-
D-cysteine
D
D
anine was observed after hydrolysis for 19 h. It was reported that
for peptides containing both oxazole and thiazole heterocycles
direct hydrolysis under acidic conditions results in the exclusive
cleavage of the oxazole ring.8,11 In order to liberate the amino acid
adjacent to thiazole ring, it is necessary to cleave the ring by ozon-
olysis prior to hydrolysis.8,11 Ozonolysis12 of 1 led to degradation of
Figure 2. Thiazole containing amino acid fragments, Ala-Tzl (2), Ala-Tzl-Phe (3)
after hydrolysis of 1.
alanylthiazole to yield
ture of hapalocyclamide (1) was established as cyclo-thiazole-
nine-oxazole- -alanine- -thiazoline- -phenylalanine.
The hapalocyclamide (1) had phytotoxic activity to suppress
lettuce (Lactuca sativa L. cv. Great Lake no. 366) seedling growth
in a concentration-dependent manner. Growth of root and shoot
was inhibited to 44.0 1.4% and 47.8 3.1% against controls,
L
-alanine. Based on these findings the struc-
with derivatized authentic amino acids. Compound 2 was identified
by MS and NMR analyses as an alanine-thiazole residue (Ala-Tzl res-
idue) (m/z 228 [M+]). This finding was confirmed by spectral com-
parison with the reported compound.7 Compound 3 (m/z 375
[M+]) contained a prominent alanine-thiazole-containing fragment
at m/z 197 (100%) as the base peak; m/z 284 (24%) derived from the
loss of 91 mass units (benzyl from Phe) and m/z 213 (38%) from the
loss of 162 mass units. These fragmentation patterns were consid-
ered to be an alanine-thiazole-phenylalanine substituent (Ala-
Tzl-Phe) (3).
L-ala-
D
D
D
NMR data (Table 1), including detailed proton decoupling
experiments, confirmed the presence of phenylalanine, two ala-
nine residues as well as a three modified amino acids: oxazole, thi-
azole, and thiazoline. The latter two modified amino acids were
derived from cysteine, indicating that there were six amino acids
in the compound. Confirmation of the sequence of the subunits
of 1 was provided by HMBC experiments as described below.
The first and second amino acids were identified as alanine. The
amide protons [d 8.66 (NH-1); d 7.45 (NH-2)] were correlated
through COSY and HMBC experiments to the nitrogen-bearing
methine [d 5.41 (H-5), 47.7 (C-5); d 4.97 (H-12), 44.0 (C-12)] and
methyl [d 1.70 (3H-6), 24.3 (C-6); d 1.45 (3H-13), 19.9 (C-13)]
groups. The third amino acid was deduced to be phenylalanine.
Figure 3. Key COSY and HMBC correlations establishing the spiral fused ring.

I. Koodkaew et al. / Tetrahedron Letters 53 (2012) 977–979
979
respectively after 48 h exposure to hapalocyclamide at 4.2
on a filter paper. Swelling of the root tips could also be observed.
50 (dose required to cause a 50% reduction in plant growth) values
l
g/cm2
were harvested by filtration and oven-dried at 60 °C for 48 h. The dried algae
were ground into a powder and kept in a desiccator until extraction and
analysis.
I
6. Acid hydrolysis:
A solution of 44.6 mg of 1 (obtained from 200 g of dry
cyanobacterium) in 1 ml of MeOH and 3 ml of 3 N HCl was heated at 100 °C
overnight. The solvent was removed under reduced pressure. The acid
hydrolysate was treated with 5% HCl in MeOH (Wako Pure Chemical
Industries, Japan). After being heated at 100 °C for 1 h, the solvent was
evaporated. The product was then reacted with Ac2O and pyridine for 24 h to
produce N-acetylated methyl ester products for GC–MS analysis. The residue
was subjected to sequential fractionation using a SiO2 column, a reversed phase
column, and preparative TLC to afford thiazole containing amino acid residues
(2, 3).
for root and shoot growth were 1.08 and 1.36 mM, respectively.
Our current research on the mode of action of hapalocyclamide
suggests that it acts on the process of mitosis.
Acknowledgments
We express our gratitude to Assistant Professor Dr. Kaori Yoko-
tani-Tomita, University of Tsukuba for her help in analyzing the
absolute stereochemistry by LC–MS. The authors are grateful to
the Chemical Analysis Center, University of Tsukuba, for NMR
experiments.
7. Wang, W.; Joyner, S.; Khoury, K. A. S.; Dömling, A. Org. Biomol. Chem. 2010, 8,
529–532.
8. Ogino, J.; Moore, R. E.; Patterson, G. M. L.; Smith, C. D. J. Nat. Prod. 1996, 59,
581–586.
9. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
10. Marfey’s analysis: Two batches of 1 (0.2 mg each) were treated with 6 N HCl
(200
HCl in vacuo, the hydrolyzate was resuspended in 40
with solution of 1-fluoro-2,4-dinitrophenyl-5- -alanine amide (FDAA,
Marfey’s reagent, 2.8 mol) in acetone (80 L) and solution of 0.1 M
NaHCO3 (20 L) in a sealed vial at 40 °C for 1 h. The reaction mixture was
quenched with 2 N HCl (10 L). The derivatized amino acids from hydrolysis
products were compared with similarly derivatized standard amino acids
(2.0 mol) by LC–MS analysis. LC separation was performed on a C18 column
(Waters, SymmetryÒ C18 column, 3.5
m, 2.1 ꢂ 150 mm) and monitored with
l
L) in sealed vials at 100 °C for 1 h and 19 h, respectively. After removal of
l
L of H2O and treated
Supplementary data
a
L
l
l
a
Color photomicrograph of Hapalosiphon sp., MS and NMR data
of compound 2 and 3, 1H and 13C NMR spectra of 1, LC–MS analysis
of Marfey’s derivatized hydrolysate of 1, details of phytotoxic
assay.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.12.048.
l
l
l
l
both UV detection at k 340 nm and ESI-MS. A linear gradient elution was
programed using a mixture of aqueous 0.01% formic acid (solvent A) and
CH3CN (solvent B) as follows; 0–30 min, 70–40% (A in B); 30–40 min, 40–20%
(A in B); 40–50 min, 20% (A in B) at a flow rate of 0.2 mL/min. One microliter of
the above prepared samples was injected and each of the amino acid FDAA
derivatives was checked by ESI-MS; alanine-FDAA [m/z 342 (M++H, 100%)];
phenylalanine-FDAA [m/z 418 (M++H, 100%)]; cysteine-bis-FDAA [m/z 626
(M++H, 32%), m/z 251 (35%), m/z 101 (100%)]. Retention times for the
References and notes
1. Burja, A. M.; Banaigs, B.; Abou-Mansour, E.; Burgess, J. G.; Wright, P. C.
Tetrahedron 2001, 57, 9347–9377.
2. Welker, M.; Döhren, H. FEMS Microbiol. Rev. 2006, 30, 530–563.
3. Sivonen, K.; Leikoski, N.; Fewer, D. P. Appl. Microbiol. Biotechnol. 2010, 86, 1213–
1225.
derivatized amino acid standards were as follows:
alanine, 11.19 min; -phenylalanine, 18.36 min; -phenylalanine, 22.31 min;
cysteine, 30.26 min;
L
-alanine, 9.36 min;
D
-
-
L
D
L
D
-cysteine, 34.57 min. In all runs, a peak at 8.17 min was
4. Hapalocyclamide:
MeOH); DI-EIMS (70 eV) m/z (rel. int.) 538 (M+, 86), 447 (100), 252 (31), 188
(25), 138 (33); HRMS m/z 538.1471 (M+, C25H26N6O4S2,
1.4 mmu), m/z
): 247 (3.91),
A
colorless needle-like crystals.
½ ꢁ ꢀ0.071° (c 0.47,
a 2D5:2
observed as excess FDAA.
11. Linington, R. G.; González, J.; Ureña, L.-D.; Romero, L. I.; Ortega-Barría, E.;
Gerwick, W. H. J. Nat. Prod. 2007, 70, 397–401.
D
12. Ozonolysis: The sample of
1 (2.0 mg) was dissolved in 4.0 mL of
447.0905 (C18H19N6O4S2,
D
ꢀ0.4 mmu); UV (MeOH) kmax nm (loge
IR (CCl4, NaCl) vmax cmꢀ1: 3398 (m), 1675 (s), 1520 (s). For 1H and 13C NMR
data, see Table 1.
dichloromethane (CH2Cl2) in a reaction vial and cooled with an ice-NaCl
bath. A stream of ozone was bubbled into the sample solution for 10 min.
Afterwards a stream of O2 was bubbled followed by N2 stream to remove
excess ozone. After the reaction was completed, CH2Cl2 was removed under a
stream of N2.
5. Hapalosiphon sp. was cultured in BG-11 liquid medium (pH 9.0) with constant
aeration in a incubator at 25 °C with continuous illumination (66–69 lmol
photon mꢀ2 sꢀ1) from a cool-white fluorescent lamp (FL20SS-W/18; National,
FL40SS-W/37; Toshiba, Japan) in a growth chamber. After 21 days, the algae