Reniochalistatins A-E, cyclic peptides from the marine sponge Reniochalina stalagmitis

Article abstract of DOI:10.1021/np5006778

Five new cyclic peptides (including four heptapeptides and one octapeptide), reniochalistatins A-E (1-5), were isolated and characterized from the marine sponge Reniochalina stalagmitis collected off Yongxing Island in the South China Sea. Their structures were assigned on the basis of HRESIMS, 1D and 2D NMR spectroscopic data, and MALDI-TOF/TOF data for sequence analysis. The absolute configurations of all of the amino acid residues were determined using chiral-phase HPLC and Marfey's analysis. The cyclic octapeptide reniochalistatin E showed biological activity in various cytotoxicity assays employing different tumor cell lines (RPMI-8226, MGC-803, HL-60, HepG2, and HeLa).

Full text of DOI:10.1021/np5006778

Article
pubs.acs.org/jnp
Reniochalistatins A−E, Cyclic Peptides from the Marine Sponge
Reniochalina stalagmitis
Kai-Xuan Zhan,†,§ Wei-Hua Jiao,‡ Fan Yang,‡ Jing Li,‡ Shu-Ping Wang,‡ Yu-Shan Li,*,† Bing-Nan Han,*,‡
and Hou-Wen Lin*,†,‡,§
†School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
‡Marine Drugs Research Center, Department of Pharmacy, State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital,
School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, People’s Republic of China
§Department of Pharmacy, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003,
People’s Republic of China
S
* Supporting Information
ABSTRACT: Five new cyclic peptides (including four
heptapeptides and one octapeptide), reniochalistatins A−E
(1−5), were isolated and characterized from the marine
sponge Reniochalina stalagmitis collected off Yongxing Island in
the South China Sea. Their structures were assigned on the
basis of HRESIMS, 1D and 2D NMR spectroscopic data, and
MALDI-TOF/TOF data for sequence analysis. The absolute
configurations of all of the amino acid residues were
determined using chiral-phase HPLC and Marfey’s analysis.
The cyclic octapeptide reniochalistatin E showed biological
activity in various cytotoxicity assays employing different tumor
cell lines (RPMI-8226, MGC-803, HL-60, HepG2, and HeLa).
of Yongxing Island in the South China Sea. Herein we report
the isolation, structure elucidation, and antitumor activities of
reniochalistatins A−E (1−5).
s sessile animals, marine sponges rely on chemical defense
Aas a major adaptation strategy to thrive in the extreme
environmental conditions of the sea, which may account for the
diverse array of bioactive chemicals that they produce.1 More
than 5300 chemically diverse compounds have been identified
from sponges including unusual nucleosides, terpenes, sterols,
cyclic peptides, alkaloids, fatty acids, peroxides, and polyke-
tides.1,2 Among them, cyclic peptides composed of both
standard and unusual amino acids have generated considerable
interest in the biomedical and pharmaceutical communities1,3
due to their antiviral, antitumor, antimicrobial, and general
cytotoxic properties. A noteworthy class of marine cyclic
peptides is represented by proline-rich heptapeptides or
octapeptides, with the presence of 2−4 proline residues, an
array of apolar residues such as Leu, Ile, and Val, and some
aromatic residues such as Phe, Tyr, and Trp.4 It has been
implied that proline residues offer conformational rigidity to
these proline-rich cyclic peptides by restricting the dihedral
angles in proline.5,8a Species of the sponge genera Phakellia,6
Axinella,7 Callyspongia,8 and Stylissa9 have been reported to
yield bioactive cyclic peptides, with most of these being rich in
proline amino acid residues. In our screening program to search
for bioactive cyclic peptides from the marine sponges of the
South China Sea, we have examined and identified the presence
of cyclic peptides rich in proline residues from an extract of a
tropical marine sponge, Reniochalina stalagmitis Lendenfeld
(class Demospongiae, order Halichondria, family Axnellidae),
RESULTS AND DISCUSSION
■
The frozen specimen of R. stalagmitis was exhaustedly extracted
with CH2Cl2/MeOH (1:1) to give an organic extract that was
partitioned between EtOAc and H2O. The EtOAc-phase extract
was subjected to solvent partitioning to yield a petroleum ether-
soluble extract and a CH2Cl2-soluble extract. Five cyclic
peptides, reniochalistatins A−E (1−5), were obtained from
the cytotoxic CH2Cl2-soluble extract by a series of chromato-
graphic separations employing Sephadex LH-20, silica gel,
ODS, and reversed-phase HPLC column chromatography.
The molecular formula of reniochalistatin A (1) was
determined as C37H62N8O8 on the basis of HRESIMS and
NMR spectroscopic analysis (Table 1). The peptidic nature of
1 was readily inferred by the presence of eight amide carbonyl
resonances, as well as seven typical amide NH resonances in
1
the H and 13C NMR spectra. Additionaly, the 13C NMR
spectrum exhibited seven α-methine carbons, which were
assigned to seven methine proton signals in the HMQC
spectrum. Detailed analysis of the HMQC, COSY, HMBC, and
TOCSY spectra allowed the identification of seven amino acid
Received: August 31, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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ions at m/z 635.0, 521.7, 424.6, 311.5, and 211.3,
corresponding to the successive loss of Leu, Pro, Ile, Val, and
the terminal dipeptide ion Pro-Asn (Figure 2).12a,13 The
ΔδCβ‑Cγ values of the Pro residues (4.7 and 9.4 ppm for Pro1
and Pro2, respectively) were indicative of a trans and a cis
geometry for the proline amide bonds in 1.9b,14
High-resolution ESIMS analysis of reniochalistatin B (2)
revealed an [M + Na]+ ion (m/z 866.4798) consistent with a
molecular formula of C46H65N7O8, thus requiring 18 degrees of
unsaturation. It was also identified as a cyclic heptapeptide on
the basis of the presence of seven carbonyl resonances and
seven α-methine carbon signals in the 13C NMR spectrum
(Table S1). Extensive inspection of the HMQC, COSY,
HMBC, and TOCSY spectra allowed the identification of the
seven amino acid residues as Pro (2×), Leu (2×), Ile, Phe, and
Tyr, of which the Pro (m/z 70.2), Phe (m/z 120.2), and Tyr
(m/z 136.2) residues were indicated by their immonium and
related ions in the MALDI-TOF/TOF spectrum (Figure
S18).12 The existence of two fragments, Pro1-Ile-Phe-Tyr-
Leu1 and Pro2-Leu2, was evidenced by the HMBC correlations
of Ile-NH/Pro1-CO and Phe-NH/Ile-CO, as well as the
NOESY correlations of Ile-NH/Pro1-Hα, Phe-NH/Ile-Hα,
Phe-NH/Tyr-Hα, Tyr-NH/Leu1-Hα, and Leu2-NH/Pro2-Hα,
respectively (Figure 1 and Table S1). Furthermore, in the
MALDI-TOF/TOF mass analysis (Figure S18), two pairs of a/
b fragment ion peaks were predominantly observed, which
covered the whole structure of 2. As a result, the connections of
the two fragments as well as the amino acid sequence of cyclic
peptide 2 cyclo-(Pro1-Ile-Phe-Tyr-Leu1-Pro2-Leu2) were con-
firmed by the MALDI-TOF/TOF sequence analysis (Figure
2).12a,13 The ΔδCβ‑Cγ values of the Pro residues (8.9 and 3.9
ppm for Pro1 and Pro2, respectively) were indicative of a cis and
a trans geometry for the respective proline amide bonds in
2.9b,14
Reniochalistatin C (3) was isolated as a colorless, glassy,
amorphous solid. The molecular formula of 3 was determined
to be C49H63N7O8 on the basis of the [M +Na]+ ion at m/z
900.4620 in the HRESIMS spectrum and confirmed by NMR
residues as Pro (2×), Leu (2×), Ile, Val, and Asn. Eight
carbonyls and the two proline rings accounted for 10 of the 11
degrees of unsaturation implied by the molecular formula,
which suggested 1 was a cyclic peptide and further supported
by the NOESY experiment (Figure 1) as well as MALDI-TOF/
TOF sequence analysis (Figure 2).
1
spectroscopic analysis. The H NMR spectrum of 3 exhibited
α- and amide protons, implying its peptide nature. The 13C
NMR spectrum revealed resonances consistent with seven
amide carbonyls and seven α-methine carbons, suggesting a
heptapeptide (Table S2). A comprehensive interpretation of
the 2D NMR spectra (COSY, TOCSY, HMQC, and HMBC)
of 3 indicated seven amino acid units: Pro (2×), Phe (2×), Ile
(2×), and Tyr. The key HMBC correlations of Phe2-NH/Tyr-
CO, as well as the NOESY correlations of Ile1-NH/Pro1-Hα,
Pro1-Hα/Phe2-Hα, Phe2-Hα/Tyr-NH, Tyr-NH/Ile2-NH, Ile2-
NH/Pro2-Hα, and Pro2-Hα/Phe1-NH, outlined the linear
amino acid sequence as Phe1-Pro2-Ile2-Tyr-Phe2-Pro1-Ile1
(Figure 1). Furthermore, the MALDI-TOF/TOF mass analysis
of 3 (Figures 2 and S31) supported the connection of Phe1 and
Ile1, which confirmed the structure of 3 as cyclo-(Pro1-Ile1-Phe1-
Pro2-Ile2-Tyr-Phe2).12a,13 The ΔδCβ‑Cγ values of the Pro
residues (7.7 and 4.4 ppm for Pro1 and Pro2, respectively)
were indicative of a cis and a trans geometry for the respective
proline amide bonds in 3.9b,14
Two fragments, Pro1-Asn-Val and Leu1-Leu2, were outlined
by the HMBC correlations of Asn-Hα/Pro1-CO, Val-NH/Asn-
CO, and Leu2-NH/Leu1-CO, respectively (Figure 1). The
NOESY correlations between Leu2-Hα/Pro1-Hδ further
allowed the connection of the fragments of Leu1-Leu2 and
Pro1-Asn-Val. The NOESY correlations between Ile-NH/Pro2-
Hδ revealed the Ile-Pro2 fragment. However, due to the
significant overlap of two carbonyl signals (Pro2, Val) in the 13C
NMR spectrum (δC 170.21 and 170.25), the connectivities of
Pro2/Leu1 and Val/Ile could not be unambiguously assigned by
the observed HMBC correlations of Pro2-Hα/Pro2-CO (δC
170.21 or 170.25) and Val-Hα/Val-CO (δC 170.21 or 170.25)
(Table 1 and Figure 1). Ultimately, the sequence of 1 was
confirmed by MALDI-TOF/TOF sequence analysis. Although
there was more than one possible ring-opening position for the
cyclic peptide, the preferred ring-opening of 1 occurred at the
Leu2-Pro1 amide bond due to the high protonation affinity
associated with the proline nitrogen (Supporting Information,
Figure S4).10,11 The immonium and related ions indicated the
existence of Leu or Ile (m/z 86.2) and Pro (m/z 70.2)
residues.12 A linearized peptide 1, Pro1-Asn-Val-Ile-Pro2-Leu1-
Leu2, was demonstrated by a main series of adjacent bn(+1)
Reniochalistatin D (4) was obtained as a colorless, glassy,
amorphous solid, and its molecular formula C48H59N7O7 was
established from the positive ion HRESIMS peak (m/z
868.4370, [M + Na]+) and the NMR data. Examination of
the COSY, TOCSY, HMQC, and HMBC spectra allowed the
identification of seven amino acid residues as Pro (3×), Phe
B
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a
1
Table 1. H (600 MHz) and 13C NMR (150 MHz) Data for Reniochalistatin A (1) in DMSO-d6
position
δC, mult
δH (J in Hz)
position
δC, mult
δH (J in Hz)
0.74, d (7.2)
Pro1
CO
α
δ
11.0, CH3
16.0, CH3
170.9, C
62.8, CH
30.0, CH2
β-Me
NH
Pro2
CO
α
0.80, d (7.2)
7.83, d (9.6)
4.01, dd (9.2, 8.0)
a: 2.26, m
β
b
b: 1.55, m
170.25, C
γ
25.3, CH2
47.8, CH2
a: 1.85, m
60.5, CH
30.9, CH2
4.24, d (7.8)
a: 2.30, m
b: 1.97, m
a: 1.85, m
b: 1.47, m
a: 3.42, m
b: 1.78, m
β
δ
a: 3.73, m
b: 3.42, m
γ
21.5, CH2
46.1, CH2
Asn
CO
α
170.6, C
49.3, CH
36.3, CH2
172.2, C
δ
4.63, dd (10.3, 5.4)
2.79, m
b: 3.26, dd (10.2, 10.2)
β
Leu1
CO
α
γ-CO
α-NH
γ-NH2
171.8, C
50.3, CH
39.9, CH2
7.81, d (6.0)
a: 7.92, s
4.39, m
β
a: 1.55, m
b: 1.16, m
1.41, m
b: 7.17, s
Val
CO
α
γ
24.6, CH
23.2, CH3
22.5, CH3
b
170.21, C
δ
0.86, d (6.6)
0.78, d (7.0)
7.40, d (7.2)
61.5, CH
29.5, CH
19.8, CH3
18.4, CH3
3.83, dd (8.0, 7.5)
2.20, m
δ′
β
NH
Leu2
CO
α
γ
0.89, d (7.2)
0.90, d (7.2)
7.99, d (8.4)
γ′
171.2, C
51.3, CH
38.3, CH2
NH
Ile
CO
α
4.14, m
β
a: 1.55, m
b: 1.18, m
1.72, m
170.0, C
57.7, CH
35.3, CH
24.9, CH2
4.10, d (9.6, 7.2)
1.92, m
γ
24.3, CH
23.6, CH3
20.9, CH3
β
δ
0.80, d (6.3)
0.86, d (6.6)
8.59, d (5.4)
γ
a: 1.31, m
δ′
NH
b: 1.07, m
a
1
Assignments of the 13C and H signals were made on the basis of DEPT, COSY, TOCSY, HMQC, HMBC, and NOESYspectroscopic data; data
b
reported in ppm. Assignments may be interchanged.
(3×), and Ile (Table S3). The HMBC correlations of Phe3-
NH/Ile-CO and Phe1-NH/Pro1-CO established Phe3-Ile and
Phe1-Pro1 fragments. Due to the significant overlap of two
carbonyl signals (δC 169.92 and 169.98 for Phe2 or Pro2) in the
13C NMR spectrum (Table S3) and the lack of the insightful
HMBC correlations of the mutiple proline residues (Figure 1),
the connectivity for each amino acid was mainly established by
the NOESY correlations of 4 (Figure 1 and Table S3).
Furthermore, the MALDI-TOF/TOF mass analysis (Figure
S44) confirmed the structure of 4 as cyclo-(Pro1-Phe1-Pro2-
Phe2-Ile-Phe3-Pro3).12a,13 The ΔδCβ‑Cγ values of the Pro
residues (9.7, 9.2, and 9.5 ppm for Pro1, Pro2, and Pro3,
respectively) were indicative of cis geometries for all proline
amide bonds in 4.9b,14
Reniochalistatin E (5) showed a major adduct ion peak at m/
z 938.5473 [M + Na]+ in the HRESIMS spectrum,
corresponding to a molecular formula of C49H73N9O8 (18
degrees of unsaturation). The 13C NMR spectrum showed
eight carbonyl peaks at δC 170.0−172.3, thus leaving
unaccounted 10 additional double bonds or rings in the
molecule (Table S4). The HMQC correlations between eight
α-proton signals and eight α-carbon resonances indicated that 5
was a peptide containing eight amino acid residues (Table S4).
Detailed analysis of the 2D NMR data obtained from COSY,
TOCSY, HMQC, and HMBC experiments revealed the
presence of Val, Leu, Ile (2×), and Pro (3×) residues,
accounting for three more degrees of unsaturation. Moreover,
in the COSY and TOCSY spectra, a downfield NH resonance
at δH 10.78 (1H, d, J = 2.7 Hz, H-1) correlated with an
aromatic proton signal at δH 6.98 (1H, d, J = 2.7 Hz, H-2),
along with the continuous spin correlations of proton signals
characteristic of a 1,2-disubstituted benzene at δH 7.47 (1H, d, J
= 10.1 Hz, H-4), 6.96 (1H, m, H-5), 7.05 (1H, t, J = 10.1 Hz,
H-6), and 7.32 (1H, d, J = 10.1 Hz, H-7), indicated the
presence of an indole ring, adding six more degrees of
unsaturation. Subsequently, long-range correlations from the β-
methylene protons at δH 3.61 and 3.37 to the carbon
resonances at δC 56.8 (Trp-Cα), 111.5 (Trp-C-3), 124.1
(Trp-C-2), and 127.3 (Trp-C-9) allowed us to assign the eighth
residue as tryptophan (Table S4). The key HMBC correlations
of Ile2-NH/Pro3-CO, Ile1-NH/Val-CO, Val-NH/Pro2-CO, and
Leu-NH/Pro1-CO, as well as the NOESY correlations of Pro3-
Hα/Ile1-Hα, Val-NH/Pro2-Hα, Pro2-Hγb/Trp-H-2, and Leu-
NH/Pro1-Hα, displayed the existence of the two fragments Ile2-
Pro3-Ile1-Val-Pro2-Trp and Pro1-Leu (Figure 1). However, due
to the significant overlap of two carbonyl signals of Ile2 and Leu
(δC 171.84 and 171.98) in the 13C NMR spectrum, the
connectivities of Leu/Trp could not be unambiguously assigned
on the basis of the observed HMBC correlations of Trp-NH/
Leu-CO (δC 171.84 or 171.98). Ultimately, the MALDI-TOF/
TOF sequence analysis was used to elucidate the connections
of the fragments (Figure S57). A linearized peptide, Pro1-Leu-
Trp-Pro2-Val-Ile1-Pro3-Ile2, was indicated by a series of adjacent
bn(+1) ions, which suggested that peptide 5 was cyclo-(Pro1-
C
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Figure 1. Key correlations of HMBC (solid arrow) and NOESY
(dashed arrow) experiments for reniochalistatins A−E (1−5).
Figure 2. MALDI-TOF/TOF sequence ions (m/z) for the protonated
molecules [M + H]+ of reniochalistatins A−E (1−5).
Leu-Trp-Pro2-Val-Ile1-Pro3-Ile2), and the preferred ring-open-
ing occurred at the Ile2-Pro1 amide bond (Figure 2).12a,13 Thus,
the macrocyclic structure was completed, accounting for the
final degree of unsaturation of reniochalistatin E (5). The
ΔδCβ‑Cγ values of the Pro residues (4.8, 4.3, and 4.1 ppm for
Pro1, Pro2, and Pro3, respectively) were indicative of trans
geometries for all proline amide bonds in 5.9b,14
A chiral-phase HPLC analysis of the hydrolyzed amino acids
of the reniochalistatins A−E (1−5) clarified that all of the Pro,
Phe, Val, Leu, Ile, and Tyr residues were L-form amino acids
(Figures S13, S27, S40, S53, and S66). Marfey’s analysis was
used to confirm the L-form of the Asn and Trp residues
(Figures S14 and S67).
Compared to the previously reported proline-rich cyclic
peptides, such as phakellistatins,6 euryjanicins,4a stylissamides,15
stylopeptides,16 and stylisins,9a reniochalistatins C (3) and D
(4) are closely related to phakellistatin 18 and stylissamide C,
with over 70% similarity in terms of peptide sequences.
Interestingly, reniochalistatins A−E (1−5) share less than 50%
similarity within the sequences. In the present work,
reniochalistatins A−E (1−5) were tested againist five different
human cancer cell lines (RPMI-8226, MGC-803, HL-60,
HepG2, and HeLa). Surprisingly, the four heptapeptides (1−
4) showed no cytotoxicity against the tested cell lines.
However, the octapeptide (5) exhibited cytotoxic activity
against myeloma RPMI-8226 and gastric MGC-803 cells with
IC50 values of 4.9 and 9.7 μM, respectively, but with no activity
against leukemia HL-60 and hepatoma HepG2 (IC50 > 20.0
μM) and cervical HeLa (IC50 17.3 μM) cells. It is premature to
draw any general conclusions regarding a structure−activity
relationship among the proline-rich cyclic peptides, owing to
conflicting reports of naturally occurring, proline-rich cyclic
peptides that were initially described as having cytotoxic
activity, but subsequent synthetic samples were not
active.5c,9a,17 This study extends our knowledge of secondary
metabolites from the marine sponges of the genus Reniochalina
previously dominated by acetylenic alcohols, dihydrothiopyr-
anone, and fatty acids.8a,18 Questions of whether these cyclic
peptides are endogenous secondary metabolites of the sponge
or its symbionts have drawn attention in the marine natural
products community.7b,19
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation measure-
ments were conducted on an Autopol I polarimeter (No. 30575,
manufactured by Rudolph Research Analytical) with a 10 cm length
cell at room temperature. UV and IR (KBr) spectra were recorded on
a Hitachi U-3010 spectrophotometer and Jasco FTIR-400 spectrom-
1
eter, respectively. H, 13C, DEPT, COSY, HMQC, HMBC, TOCSY,
D
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and NOESY NMR spectra were acquired at room temperature on
Bruker AVANCE-600 and Bruker AMX-500 instruments with DMSO-
d6 as the solvent. Spectra were referenced to residual solvent signals
with resonances at δH 2.49 and δC 39.7. High-resolution ESIMS
spectra were acquired with a Waters Q-Tof micro YA019 mass
spectrometer. MALDI-TOF/TOF spectra were recorded on a 4700
Proteomics analyzer (Applied Biosystems). Reversed-phase HPLC was
performed on a YMC-Pack Pro C18 column (250 × 10 mm, 5 μm)
using a Waters 600 HPLC instrument with a Waters 996 UV detector.
Column chromatography (CC) was performed on ODS (15 μm, YMC
Co.) and Sephadex LH-20 (18−110 μm, Pharmacia Co.). The
fractions were monitored by TLC (HSGF 254, Yantai, China), and
spots were visualized by heating silica gel plates sprayed with 10%
H2SO4 in H2O. The enantioselective HPLC of the amino acids was
conducted with a chiral-phase column (MCI GEL CRS10W, 4.6 × 50
mm, Mitsubishi Chemical Corporation). Amino acid standards used
for enantio selective analysis were purchased from Sigma-Aldrich
Chemical Corporation.
Animal Material. Specimens of Reniochalina stalagmitis Lendenfeld
were collected around Yongxing Island in the South China Sea during
the month of July in 2009 and were identified by Mrs. Shirley Sorokin
(Centre of Marine Bioproducts Development, Flinders University,
Australia). A voucher sample (No. SZH) was deposited in the
Laboratory of Marine Drugs, Department of Pharmacy, Changzheng
Hospital, Second Military Medical University, China. The sponge R.
stalagmitis is of great abundance in the Xisha Islands, and therefore this
collection was considered to have no significant adverse ecological
effect.
MALDI-TOF/TOF data, Figure 2; HRESIMS m/z 868.4370 [M +
Na]+ (calcd for C48H59N7O7Na, 868.4374).
Reniochalistatin E (5, cyclo-(L-Pro-L-Leu-L-Trp-L-Pro-L-Val-L-Ile-L-
Pro-L-Ile)): colorless, glassy, amorphous solid; [α]25 −100 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 221 (4.38), 283 (3.61), 291 (3.55)
nm; IR (KBr) νmax 3292, 2962, 2931, 1681, 1623, 1520, 1449 cm−1; 1H
and 13C NMR data, Table S4; MALDI-TOF/TOF data, Figure 2;
HRESIMS m/z 938.5473 [M + Na]+ (calcd for C49H73N9O8Na,
938.5480).
Chiral-Phase HPLC Analysis of the Acid Hydrolysate of 1−5.
Each of the peptides 1−5 (0.5 mg) was dissolved in 6 N HCl (1 mL)
and heated at 110 °C for 18 h. After cooling to room temperature (rt),
the solvent was evaporated, and traces of HCl were removed by
repeated drying under vacuum with distilled H2O. The dried
hydrolysate was dissolved in 500 μL of 2 mM CuSO4/H2O solution.
The hydrolysates of peptides 1−5 and authentic L- and D-amino acids
were analyzed with a chiral-phase column (MCI GEL CRS10W, 4.6 ×
50 mm, Mitsubishi Chemical Corporation) using 2 mM CuSO4/H2O
solution as the mobile phase with UV detection at 254 nm on a Waters
1525/2998 HPLC instrument. All amino acid residues in reniocha-
listatins A−E (1−5) were found to correspond to the L-configuration
by comparison of retention time values (tR, min) with those of
standard amino acids: (1) aqueous 2 mM CuSO4, flow rate at 1 mL/
min, L-Ile (19.9), D-Ile (8.7), L-allo-Ile (14.2), D-allo-Ile (7.6), L-Leu
(17.3), D-Leu (9.2), L-Tyr (28.2), D-Tyr (16.1), L-Pro (6.2), D-Pro
(3.3), L-Phe (45.7), D-Phe (25.8); (2) aqueous 2 mM CuSO4, flow rate
at 0.8 mL/min, L-Ile (26.2), D-Ile (10.9), L-allo-Ile (18.6), D-allo-Ile
(10.4), L-Leu (23.0), D-Leu (12.1), L-Pro (7.9), D-Pro (4.2), L-Val
(9.7), D-Val (5.2) (Figures S13, S27, S40, S53, and S66).
Extraction and Purification. The extracts from the sponge (72 g,
dry wt) were obtained with CH2Cl2/MeOH (1:1), and combined
extracts were concentrated under vacuum. The concentrated extract
was then suspended in H2O, and EtOAc was used to extract the
organic components. The EtOAc-soluble extract was partitioned
between MeOH/H2O (9:1) and petroleum ether to afford the
petroleum ether-soluble extract (1.1 g). The MeOH/H2O (9:1) phase
was diluted to 3:2 with water, which was partitioned by CH2Cl2 to
yield the CH2Cl2-soluble extract (1.3 g). This CH2Cl2-soluble extract
was subjected to CC on Sephadex LH-20 using CH2Cl2/MeOH (1:1)
as eluent to give three subfractions (A−C). Subfraction B (700 mg)
was subjected to CC on ODS silica and further purified by HPLC
(YMC-Pack Pro C18, 5 μm, 10 × 250 mm, 2.0 mL/min, UV detection
at 210 and 275 nm) eluting with CH3CN/H2O (50:50) to yield pure
peptides reniochalistatin B (2, 9.0 mg, tR 40.9 min) and
reniochalistatin D (4, 5.1 mg, tR 49.7 min); CH3CN/H2O (45:55)
to yield pure reniochalistatin A (1, 5.8 mg, tR 17.1 min); CH3CN/H2O
(60:40) to yield pure reniochalistatin C (3, 3.0 mg, tR 26.6 min); and
CH3CN/H2O (55:45) to yield reniochalistatin E (5, 2.8 mg, tR 29.2
min).
HPLC Analysis of the Acid Hydrolysates of 1 and 5 Using
Marfey’s Method. To determine the absolute configurations of the
Asn in 1 and the Trp in 5, HPLC analyses of the acid hydrolysates
were conducted. Compounds 1 (0.36 mg) and 5 (0.50 mg) were
hydrolyzed as mentioned above. The hydrolysate was processed using
a published protocol20 with the following exceptions: (1) 20 μL of 1 M
HCl was used; (2) the resulting residue after solvent removal was
resuspended in 100 μL of MeOH; (3) a YMC-Park Pro C18, 4.6 × 250
mm, 5 μm column was used. Under typical acid hydrolysis conditions,
Asn residues will be converted to Asp residues.21 The retention times
for FDAA derivatives of L-Asp, D-Asp, L-Trp, and D-Trp were 11.6,
12.5, 23.6, and 24.5 min, respectively. The FDAA derivatives of the
amino acids from the hydrolysate of 1 and 5 showed peaks at 11.7 (L-
Asp) and 23.7 (L-Trp) min, respectively (Figures S14, S67), and the
amino acids were assigned to be L-Asn in 1 and L-Trp in 5.
Cell Culture. The human myeloma RPMI-8226 cell line was
provided by Dr. Jian Hou, Changzheng Hospita (Second Military
Medical University, Shanghai, China). The human gastric cancer
MGC-803 cell line, human cervical cancer HeLa cell line, human
leukemia HL-60 cell line, and human hepatoma HepG2 cell line were
purchased from the Type Culture Collection (Chinese Academy of
Sciences, Shanghai, China). The RPMI-8266 and MGC-803 cell lines
were cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum (FBS) and incubated at 37 °C in an atmosphere of 5%
CO2. The HL-60 cell line was cultured in RPMI-1640 medium
supplemented with 20% FBS and incubated at 37 °C in an atmosphere
of 5% CO2. The HeLa and HepG2 cell lines were cultured in HG-
DMEM, supplemented with 10% FBS in 5% CO2 at 37 °C.
Biological Assays. The cytotoxic activities of these comounds
were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-
thoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) assay as
described previously.22 RPMI-8266, HeLa, MGC-803, HepG2, and
HL-60 cells were seeded in 96-well microplates at a density of 1 × 104
cells/well in 100 μL of growth media and incubated for 24 h. Culture
media containing different concentrations of the samples were then
added. After incubation for 72 h, 20 μL of MTS was added to each
well, and the plate was incubated for 2 h. The absorbance was
measured spectrophotometrically at a wavelength of 490 nm (with a
reference wavelength of 690 nm) using a SpectraMax 340 plate
reader/spectrophotometer (Molecular Devices Corp.). The positive
controls were SAHA against RPMI-8226 (IC50 2.4 μM) and
Reniochalistatin A (1, cyclo-(L-Pro-L-Asn-L-Val-L-Ile-L-Pro-L-Leu-L-
Leu)): colorless, glassy, amorphous solid; [α]25D −130 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 207 (4.13) nm; IR (KBr) νmax 3312, 2959,
1
1643, 1529, 1432 cm−1; H and 13C NMR data, Table 1; MALDI-
TOF/TOF data, Figure 2; HRESIMS m/z 769.4586 [M + Na]+ (calcd
for C37H62N8O8Na, 769.4588).
Reniochalistatin B (2, cyclo-(L-Pro-L-Ile-L-Phe-L-Tyr-L-Leu-L-Pro-L-
Leu)): colorless, glassy, amorphous solid; [α]25D −110 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 208 (4.25), 279 (3.02) nm; IR (KBr) νmax
3320, 2958, 1645, 1517, 1447 cm−1; 1H and 13C NMR data, Table S1;
MALDI-TOF/TOF data, Figure 2; HRESIMS m/z 866.4798 [M +
Na]+ (calcd for C46H65N7O8Na, 866.4792).
Reniochalistatin C (3, (cyclo-(L-Pro-L-Ile-L-Phe-L-Pro-L-Ile-L-Tyr-L-
Phe)): colorless, glassy, amorphous solid; [α]25D −110 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 212 (4.34), 277 (3.25) nm; IR (KBr) νmax
1
3342, 3244, 2964, 2931, 1653, 1622, 1516, 1446 cm−1; H and 13C
NMR data, Table S2; MALDI-TOF/TOF data, Figure 2; HRESIMS
m/z 900.4620 [M + Na]+ (calcd for C49H63N7O8Na, 900.4636).
Reniochalistatin D (4, cyclo-(L-Pro-L-Phe-L-Pro-L-Phe-L-Ile-L-Phe-L-
Pro)): colorless, glassy, amorphous solid; [α]25 −66 (c 0.1, MeOH);
D
UV (MeOH) λmax (log ε) 212 (4.29) nm; IR (KBr) νmax 3275, 2965,
1646, 1550, 1446, 1431 cm−1; 1H and 13C NMR data, Table S3;
E
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Journal of Natural Products
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ASSOCIATED CONTENT
* Supporting Information
■
S
Copies of 1D and 2D NMR, HRESIMS, UV, IR, MALDI-
TOF/TOF spectra, and chiral HPLC and Marfey’s analysis for
compounds 1−5. These materials can be accessed free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*(Y. S. Li) E-mail: lyshan888@126.com.
*(B. N. Han) E-mail: hanbingnan@shsmu.edu.cn.
*(H. W. Lin) Tel: +86-21-58732594. Fax: +86-21-68383346.
E-mail: franklin67@126.com.
Notes
The authors declare no competing financial interest.
(8) (a) Ibrahim, S. R. M.; Min, C. C.; Teuscher, F.; Ebel, R.;
Kakoschke, C.; Lin, W.; Wray, V.; Edrada-Ebel, R.; Proksch, P. Bioorg.
Med. Chem. 2010, 18, 4947−4956. (b) Ibrahim, S. R. M.; Edrada-Ebel,
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ACKNOWLEDGMENTS
■
We would like to thank Mrs. S. Sorokin (Centre of Marine
Bioproducts Development, Flinders University, Australia) for
identifying the sponge. This research was supported by the
National Natural Science Fund for Distinguished Young
Scholars of China (81225023), the National Natural Science
Fund of China (Nos. 41476121, 81402844, 81302691,
81373321, 41106127, 81172978, 81072573, and 81001394),
and Shanghai Subject Chief Scientist (12XD1400200). We are
also grateful for the financial support of the National High
Technology Research and Development Program of China
(863 Projects, Nos. 2013AA092902 and 2011AA09070107).
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