Structures and solution conformational dynamics of stylissamides G and H from the Bahamian Sponge Stylissa caribica

Article abstract of DOI:10.1021/np400891s

Two new peptides, stylissamides G and H, were isolated from extracts of a sample of Stylissa caribica collected in deep waters of the Caribbean Sea. A single sample of S. caribica among a collection of 10 samples that were examined by LC-MS appeared to be a different chemotype from the others in that it lacked the familiar pyrrole-2-aminoimidazole alkaloids, stevensine and oroidin, and contained peptides of the stylissamide class. The structures of the title compounds were solved by integrated analysis of the MS and NMR spectra and chemical degradation. The solution conformation of stylissamide G was briefly examined by electronic circular dichroism and temperature-dependent ¹H NMR chemical shifts of amide NH signals, which supported a conformationally rigid macrocycle.

Full text of DOI:10.1021/np400891s

Article
pubs.acs.org/jnp
Structures and Solution Conformational Dynamics of Stylissamides G
and H from the Bahamian Sponge Stylissa caribica
Xiao Wang,† Brandon I. Morinaka,†,‡ and Tadeusz F. Molinski*,†,§
†Department of Chemistry and Biochemistry and §Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
S
* Supporting Information
ABSTRACT: Two new peptides, stylissamides G and H, were
isolated from extracts of a sample of Stylissa caribica collected
in deep waters of the Caribbean Sea. A single sample of S.
caribica among a collection of 10 samples that were examined
by LC-MS appeared to be a different chemotype from the
others in that it lacked the familiar pyrrole-2-aminoimidazole
alkaloids, stevensine and oroidin, and contained peptides of
the stylissamide class. The structures of the title compounds
were solved by integrated analysis of the MS and NMR spectra
and chemical degradation. The solution conformation of
stylissamide G was briefly examined by electronic circular
dichroism and temperature-dependent 1H NMR chemical
shifts of amide NH signals, which supported a conformationally rigid macrocycle.
he marine sponge Stylissa caribica Lehnert & van Soest,
045Q) collected from Sweetings Cay, with a reversed-phase
T1998 is found at depths in excess of −25 m in the LCMS profile different from other samples, lacked antifungal
activity, but showed significant cytotoxic activity toward
cultured human colon tumor cells (HCT-116). While mainly
3 and smaller amounts of 2a were evident in most samples of S.
caribica,11 these two compounds were not detected in 08-045Q
(LCMS analysis). Instead, the latter sample contained
hymenidin (2b) as the major PAI along with late-eluting
components that lacked Br and with m/z values in the range
expected for stylissamides. Sequential solvent partitioning of a
MeOH extract of the sample separated the constituents by
polarity, to give an n-BuOH-soluble fraction that was further
purified by preparative HPLC to provide a cytotoxic fraction
(IC50 = 5.5 μg·mL−1). Rechromatography of the latter gave
pure samples of two new heptapeptides, stylissamides G (1g)
and H (1h).
Caribbean Sea. The cyclic heptapeptides stylissamides A−F
(1a−d,1 e, f;2,3 see Supporting Information for structures) have
recently been described from S. caribica from the Bahamas.
Many related peptides exhibit biological activity; for example,
stylissamide ‘X’, from an unidentified Stylissa species from
Indonesia, inhibited EGF-induced migration of cultured HeLa
cells.4 The cyclic peptides are minor components of these
sponges with the major secondary metabolites being, largely,
brominated pyrrole 2-aminoimidazole alkaloids5 (PAIs) such as
oroidin (2a),6,7 hymenidin (2b),8 and stevensine (3)9 and their
higher-order oligomeric congeners.
Recently, we demonstrated de novo biosynthesis of 15N-
labeled PAIs (nagelamide H and benzosceptrin C) from 15N-
2a,10 providing the first experimental evidence that oroidin is a
biosynthetic precursor of higher-order PAIs and participates in
single-electron C−C bond forming reactions catalyzed by a
metallo-enzyme (or enzymes) with molecular oxygen as the
terminal oxidant.11 The provenance and biosynthetic origins of
the stylissamides, however, are less certain.
The molecular formula of 1g, C45H61N7O7, was established
from HRESIMS data. The high N content of 1g and
1
preliminary analysis of H NMR data (Table 1) suggested a
heptapeptide; this was further supported by the presence of
cross-peaks in the HSQC spectrum consistent with seven α-CH
signals (δ 51.6−61.6 ppm) and seven amide carbonyl signals (δ
170.6−171.7 ppm). Analysis of COSY and HMBC spectra of
1g provided assignments of spin systems due to one each of
Leu and Ile, two Phe, and three Pro residues (Table 1). All
three Pro residues could be assigned cis conformations based on
empirical rules13 that correlate with 13C NMR chemical shifts
RESULTS AND DISCUSSION
■
The secondary metabolite profile of S. caribica has been
investigated previously by HPLC.12 In our screening for
antifungal and cancer cell-inhibitory marine secondary metab-
olites, we examined several samples (N = 10) of S. caribica
collected from the Bahamas in 2007 and 2008. Most showed in
vitro antifungal activity against fluconazole-resistant Candida
albicans, Can. krusei, Cryptococcus neoformans var. grubbii, and
Cryp. neoformans var. gatti. A single sample of S. caribica (08-
Special Issue: Special Issue in Honor of Otto Sticher
Received: October 21, 2013
Published: February 27, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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a
1
Table 1. H and 13C NMR Data for 1g (DMSO-d6, 600
MHz)
position
13C, type
171.7, C
1H, mult (J in Hz)
Ile
CO
α
53.1, CH
37.1, CH
14.5, CH3
23.8, CH2
4.26, t (9.3)
1.59, m
β
βCH3
0.75, m
γ
1.98, m
1.45, m
δCH3
NH
CO
α
10.6, CH3
0.77, t (7.3)
7.88, d (9.3)
Pro1
169.6, C
61.0, CH
31.0, CH2
4.34, brd (9.1)
2.00, m
β
2.18, m
γ
21.3, CH2
1.32, m
1.72, m
δ
45.6, CH2
167.7, C
3.31, m
Phe1
CO
α
β
51.8, CH
35.2, CH2
4.37, m
3.36, m
2.98, dd (13.6, 0.6)
6.91, d (6.4)
7.22, m
Ph
129.6 (o)
127.7 (m), CH
126.4 (p), CH
135.5, C
7.21, m
NH
CO
α
6.55 brs
Pro2
171.3, C
57.1, CH
30.4, CH2
4.61, d (8.0)
2.16, m
1.92, m
1.89, m
1.81, m
3.52, m
3.31, m
β
γ
21.1, CH2
46.7, CH2
δ
Phe2
CO
168.8, C
52.9, CH
36.0, CH2
α
β
4.41, ddd (9.2, 6.4, 1.8)
3.09, dd (13.5, 6.4)
2.95, dd (13.5, 9.2)
7.28, m
(δ Cγ < 23.3 ppm) and differences between the β and γ carbons
[Δδ(Cβ−Cγ) > 8.0 ppm].
Ph
128.9 (o), CH
128.5 (m), CH
126.7 (p), CH
135.7, C
7.37, t (7.6)
Sequence assignments of 1g were obtained by analysis of
HMBC and NOESY data. A band-selective constant-time
HMBC14 experiment (ΔδC 161−181 ppm, optimized for J = 8
Hz) showed correlations from δ 4.37 (Phe1Hα) to δ 169.9
7.28, m
NH
CO
α
8.94, s
Pro3
169.9, C
60.1, CH
30.0, CH2
(Pro1CO), δ 4.34 (Pro1Hα) to δ 171.7 (IleCO), δ 7.88 (IleNH
)
3.27, m
1.89, m
1.04, m
1.62, m
1.27, m
3.27, m
3.18, t (9.8)
and δ 4.26 (IleHα) to δ 170.6 (LeuCO), δ 8.83 (LeuHα) to δ
169.9 (Pro3CO), δ 3.27 (Pro3Hα) to δ 168.8 (Phe2CO), and δ
8.94 (Phe2NH) to δ 171.4 (Pro2CO). The foregoing data were
sufficient to define the sequence Pro1-Phe1-Pro2-Phe2-Pro3-Leu-
Ile. Support for this sequence was also obtained from NOESY
data. Cross-peaks in the NOESY spectrum of 1g were observed
between the following pairs of amino acid residue NH and α-
β
γ
21.4, CH2
45.8, CH2
δ
Leu
CO
170.6, C
51.1, CH
38.0, CH2
CH NMR signals: Phe2NH/Pro2Hα, Pro2Hα/Phe1Hα, Pro1
/
Hκ
α
β
4.06, m
IleHα, IleNH/LeuHα, LeuNH/Pro3Hα, and Pro3Hα/Phe2 (Figure
Hα
1.52, m
1).
1.10, m
Stylissamide G (1g) is a variant on the heptapeptide motif
detected in cyclic peptides, from not only Stylissa (family
Dictyonellidae) but other sponges in the family Axinellidae.
Cytotoxic phakellistatins constitute a family of over a dozen
peptides characterized from different samples of Phakellia spp.
from the Indian Ocean,15 Western Pacific,16 and the South
China Sea17 mostly with the typical heptapeptide motif of
γ
24.5, CH
20.3, CH3
23.1, CH3
1.51, m
δ
0.73, d (6.3)
0.81, d (6.4)
8.83, d (7.1)
δ′
NH
a
See structures of 1g and 1h for the amino acid residue key. 13C NMR
shifts from indirect 2D NMR (HSQC, HMBC, and bsctHMBC).
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a
Table 2. 1H and 13C NMR Data for 1h (CD2Cl2, 600 MHz)
position
13C, δ, type
1H δ, mult (J in Hz)
Ile
CO
168.9, C
α
59.7, CH
35.8, CH
15.5, CH3
25.3, CH3
11.3, CH3
3.88, t (5.1)
1.39, m
β
βCH3
0.77, d (6.9)
1.16, m
γ
δ
0.63, t (7.4)
5.63, d (4.8)
NH
CO
α
Ser
Phe
Val
171.9, C
Figure 1. Selected NOESY and HMBC correlations for stylissamide G
(1g).
54.2, CH
63.22, CH2
4.75, d (9.9)
4.64, d (11.8)
3.42, d (11.8)
7.21, m
β
NH
CO
α
171.7, C
63.2, CH
36.1, CH2
stylissamides. In terms of sequence and amino acid
composition, peptide 1g most closely resembles phakellasta-
tin-2 (4)15 and stylisins-1 (5a) and -2 (5b) from S. caribica
collected in Jamaica.18
3.79, dd (15.5, 7.9)
3.67, m
β
3.53, dd (14.0, 7.9)
8.36, s
NH
The molecular formula of 1h, C44H58N8O8, was established
CO
N.D.
1
from HRESIMS data. The H NMR spectrum of cyclopeptide
α
56.0, CH
33.0, CH
17.1, CH3
20.1, CH3
4.30, m
1h exhibited conformational heterogeneity in several solvents
(CDCl3, DMSO-d6, and CD3OD) that significantly compli-
cated interpretation and assignment. Similar solvent-dependent
β
1.86, m
γ
0.87, d (6.9)
0.84, d (6.9)
8.54, d (7.7)
γ′
complexity has been observed in the NMR spectra of 4.19
A
NH
CO
α
1
relatively well-resolved H NMR spectrum of 1h was obtained
in CD2Cl2, and analysis of the spin systems by proton, COSY,
HMQC, and HMBC NMR (Table 2) showed the presence of
Trp, Ser, Ile, Val, Phe, and two Pro residues. Pro1 was assigned
the cis conformation, while Pro2 was assigned as trans according
to empirical rules. Attempts to sequence 1h by HMBC and
NOESY failed due to relatively weak carbonyl signals dispersed
between multiple conformers. Alternative MS4 analysis also
failed to deliver interpretable fragment ions. Gratifyingly, partial
hydrolysis of 1h (2 M HCl in CH3CN−H2O, 60 °C, 90 min)
gave a linear peptide from single amide bond hydrolysis that
was identified as Pro1-Trp-Pro2-Ile-Ser-Phe-Val by LC/MS/MS
(Figure 2). The composition and amino acid sequence of 1h
are identical to those of euryjanicin A (6),20 which was isolated
by Rodriguez and co-workers from the Caribbean sponge
Pro1
N.D.
61.2, CH
30.8, CH2
4.29, m
2.02, m
1.95, m
1.57, m
0.99, m
3.35, m
3.34, m
β
γ
20.9, CH2
46.5, CH2
δ
Trp
CO
N.D.
α
β
50.0, CH
27.8, CH2
5.23, m
3.29, m
3.11, dd (15.7, 7.5)
6.80, d (9.0)
NH
CO
α
Pro2
172.9, C
63.1, CH
29.7, CH2
4.26, t (8.3)
2.42, m
1.86, m
2.07, m
1.84, m
3.93, m
3.67, m
́
Prosuberites laughlini (Diaz, Alvarez & Van Soest, 1987).
β
However, peptides 1h and 6 are configurational isomers: Pro2 is
in the trans configuration in 1h, while both proline residues in 6
adopt the cis configuration. No interconversion of 1h into 6 was
observed upon heating the former in DMSO (70 °C, 30 min),
suggesting that 1h is the thermodynamically favored isomer.
The amino acid residues in all Stylissa peptides reported to
date are of the L-configuration. Pure samples of 1g and 1h were
separately hydrolyzed (6 M HCl, 95 °C, 15 h), and, after
removal of volatiles, the residues were subjected to Marfey’s
analysis.21 The configurations of all amino acid residues in both
peptides (except Trp in 1h, which decomposed during
hydrolysis) were also found to be L.22
γ
25.4, CH2
47.9, CH2
δ
a
See structures of 1g and 1h for the amino acid residue key. 13C NMR
chemical shift assignments from indirect 2D NMR (HSQC, HMBC,
and bsctHMBC.
At room temperature (23 °C), the CD spectrum of 1g
(CH3CN, Figure 3) was characterized by two Cotton effects
(CEs) (λ 196 (Δε +23.2), 223 (Δε −7.2) nm), while that of 1h
showed more complex band structure due to the presence of
Trp. At elevated temperatures (50 and 70 °C, Figure 4), little
difference was observed in the CD spectra of 1g and 1h,
although at lower temperature (0 °C) moderate increases in the
magnitudes of both CEs of 1g were observed.
Natural product cyclic heptapeptides and octapeptides are
presumed to adopt rigid, conformationally constrained
structures ordered by minimized torsional strain, especially
those imposed by cis and trans Pro residues. Aside from
computational modeling studies that determined the minimized
energy structures,18 no experimental data have been reported
on the conformational dynamics of the stylissamides. We briefly
examined the solution conformational dynamics of 1g and 1h
by temperature-dependent CD spectra (Figure 4) and amide
1
In their study of temperature-dependent H NMR amide
NH signals and exchange rates in proteins, Baxter and
Williamson concluded that temperature coefficients (Δδ/ΔT)
greater than −4.5 ppb/K were correlated with hydrogen-
1
signal (NH) H NMR chemical shifts (Table 3).
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Figure 2. LC/MS/MS analysis of the partial hydrolysis product of stylissamide H (1h).
Figure 3. CD and UV spectra (CH3CN, 23 °C): (a) stylissamide G (1g) and (b) stylissamide H (1h).
bonded amide protons, while NH protons with values less than
are strongly hydrogen bonded. These temperature coefficients
support a generally tightly constrained amide backbone for 1g
with weaker hydrogen bonding at the Phe2 and Ile residues.
Taking both CD and Δδ/ΔT measurements into consideration,
the data support conformations of 1g and 1h with relatively
−4.5 ppb/K were not hydrogen-bonded.23 Measurement of
1
temperature-dependent H NMR amide NH chemical shifts of
1g (T = 25, 50, 70 °C) gives Δδ/ΔT coefficients (Table 3) that
are greater than −4.5 ppb/K for Phe1 and Leu, but lower than
−4.5 ppb/K for Phe2 and Ile, showing that NH of Phe1 and Leu
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Figure 4. Temperature-dependent CD spectra (CH3CN): (a) stylissamide G (1g) and (b) stylissamide H (1h).
with a 1.7 mm {13C/15N}1H microcryoprobe, operating at 599.5556
Table 3. Temperature Coefficients (Δδ/ΔT) of Amide NH
1H NMR Signals (δ, parts per billion) for 1g
1
MHz. Additional H and 13C NMR spectra were measured on a Jeol
ECA 500 spectrometer operating at 500.1599 and 125.6940 MHz,
respectively, with samples dissolved in CD3OD or DMSO-d6 (99.8
atom % D, Cambridge Isotopes). Low-resolution MS spectra were
recorded on a ThermoFisher MSQ Plus Surveyor single quadrupole
mass spectrometer, operating in positive ion ESI mode, coupled to a
Thermo Fisher Accela ultra-high-performance liquid chromatograph
(UHPLC). HRMS measurements were made using an Agilent 6230
TOFMS under positive ion ESI TOFMS conditions and provided by
the UCSD Small Molecule MS Facility and the Torrey Pines Scripps
Research Institute, La Jolla, California. Semipreparative HPLC was
carried out using an Agilent 1100 HPLC under specified columns and
gradient conditions.
Animal Material, Extraction, and Isolation. A sample of Stylissa
caribica (accession 08-08-045) was collected at Sweetings Cay,
Bahamas (49.646′ S, 77°54.054′ W) using scuba from a depth of
−27 m in June 2008 and kept frozen (−20 °C) until required. The
frozen sponge (wet wt 107.1 g) was cut into ∼3 cm cubes and
extracted with 1:1 CH2Cl2−MeOH with stirring (rt, overnight, 2 ×
500 mL). Following removal of the solvent under reduced pressure,
the residue was partitioned between hexanes (3 × 250 mL) and 9:1
MeOH−H2O (250 mL). The aqueous MeOH layer was concentrated
and further partitioned between n-BuOH (3 × 250 mL) and H2O
(250 mL). Removal of the volatile solvent from the upper layer gave a
brown semisolid (1.68 g), a portion of which was adsorbed onto a
solid-phase extraction cartridge (C18) and eluted with MeOH. The
MeOH eluate was concentrated under reduced pressure to give a solid
(870 mg), which was separated by semipreparative reversed-phase
HPLC (C18, gradient from 10:90 to 60:40 CH3CN−H2O + 0.1%
TFA) to give 10 fractions. A portion (8.6 mg) of the eighth fraction
(36.8 mg) was further purified by semipreparative HPLC (C18,
gradient 34:8:58 CH3CN−iPrOH−H2O + 0.1% TFA) to give
didebromonagelamide A11 (0.4 mg). The tenth fraction (72.3 mg)
was found to be cytotoxic against HCT-116 cells (IC50 = 5.5
μg.mL−1). A portion of the tenth fraction (22 mg) was further purified
by reversed-phase HPLC (C18 Phenomenex 250 × 10 mm 5 μm, 100
Å column, 3.0 mL·min−1 under the following gradient profile (A =
H2O−0.1% CF3COOH, B = CH3CN; 40% B, t = 0 min; 40% B, t = 5
min; 60% B, t = 20 min) and gave stylissamide H (tR = 12.6 min, 0.50
mg, 1h, 0.015% wet wt), stylissamide G (1g, tR = 15.1 min, 1.6 mg,
0.049% wet wt), and known compounds stylisin-1 (5, tR = 9.78 min),
stylissamide A (1a, tR = 18.5 min), and stylissamide C (1c, tR = 18.5
T (K)
298
323
343
δ (ppm, N−H)
Δδ/ΔT (ppb/K)
Ile
7.88
8.83
6.55
8.94
7.73
8.80
6.58
8.82
7.61
8.76
6.61
8.72
−6.0
−1.5
1.3
Leu
Phe1
Phe2
−4.9
rigid amide backbones, constrained by intramolecular hydrogen
bonds, but higher mobility of the Ile and Phe residues.
Pettit and others have reported potent cytotoxicity of cyclic
heptapeptides related to 1g and 1h, for example, phakellistatin-
2,15 related phakellistatins,16 and hymenistatins G, H, and J.17
Kobayashi and co-workers showed that stylissamide ‘X’
inhibited EGF-induced migration of HeLa cells in the
concentration range 0.1 to 10 μM.4 In the present work, the
fraction of S. caribica extract containing stylissamides 1g and 1h
was cytotoxic to HCT-116 cells. Peptide 1h exhibited modest
in vitro cytotoxicity against HCT-116 (EC50 5.7 μM) while 1g
was essentially inactive (EC50 >200 μM).
In conclusion, two new heptapeptides, stylissamides G (1g)
and H (1h), were isolated from a cytotoxic fraction of the
sponge S. caribica from the Bahamas and characterized by
integrated spectroscopic methods. Studies of the solution
dynamics of 1g and 1h by temperature-dependent CD and
amide NH 1H NMR temperature coefficients suggested peptide
conformations ordered by a relatively rigid, hydrogen-bonded
backbone structure with limited mobility.
EXPERIMENTAL SECTION
General Experimental Procedures. UV−vis spectra were
recorded on a Jasco UV V630 dual beam instrument in 1 cm quartz
cells. CD spectra were recorded on a Jasco 810 spectropolarimeter in
quartz cells (1 or 2 mm path length). H NMR and 2D NMR spectra
of submilligram samples were measured on a Bruker 600 NMR
spectrometer under control of an Avance III console and equipped
■
1
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min) as colorless solids. See Supporting Information for complete
structures.
REFERENCES
■
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Stylissamide G (1g): colorless solid; UV (CH3CN, 23 °C) λmax (log
ε) 258 (2.88), 264 (2.87) nm; CD (0.322 mM, CH3CN, 23 °C) λmax
(2) Cychon, C.; Kock, M. J. Nat. Prod. 2010, 73, 738−742.
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1
(Δε) 196 (+23.2), 223 (−7.2) nm, see also Figures 3 and 4; H and
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13C NMR, see Table 1; HRESIMS m/z 812.4704 [M + H]+, 834.4522
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[M + Na]+ (calcd for C45H62N7O7 812.4705; calcd for
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(ref 14). A band-selective constant-time HMBC spectrum of a sample
of 1g (∼1.0 mg) in CDCl3 was recorded under the following
conditions: the band-selective pulse was a G3 Gaussian cascade of 500
μs duration centered at δ 172 ppm. The 13C spectral width was set to
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20 ppm, and H-detected FIDs (64 t1 increments) were collected at
600 MHz for a total experiment time of 84 min. The delay for
selection of long-range coupling was set to J = 8 Hz, and the 2-fold
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between J = 120 and 180 Hz.
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ASSOCIATED CONTENT
2383.
■
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1 (858) 534-7115. Fax: +1 (858) 822-0386. E-mail:
tmolinski@ucsd.edu.
Present Address
‡Institute of Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse
̈
10, 8093 Zurich, Switzerland.
̈
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Y. Su and B. Duggan (UCSD) for assistance with
MS-MS analysis and band-selective HMBC, respectively, J.
Kwan for preparative HPLC prepurifications and M. Choi and
M. Kashyap for cytotoxicity data. We are grateful to J. Pawlik
(University of North Carolina, Wilmington) and the crew of
the RV Seward Johnson for assistance with the logistics of
sample collection. The 500 MHz NMR spectrometer and the
HPLC TOFMS were purchased with funding from the NSF
(Chemical Research Instrument Fund, CHE0741968) and the
NIH Shared Instrument Grant (S10RR025636) programs,
respectively. B.I.M. was supported by a Ruth L. Kirschstein
National Research Service Award NIH/NCI (T32 CA009523).
Generous funding for this work was provided by grants from
the NIH (AI039987 and AI100776 to T.F.M.).
DEDICATION
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Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich,
Switzerland, for his pioneering work in pharmacognosy and
phytochemistry.
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