Proline-containing cyclopeptides from the marine sponge phakellia fusca

Article abstract of DOI:10.1021/np9008267

Four new cyclopeptides, phakellistatins 15-18 (2-5), together with five known cyclopeptides, phakellistatin 13 (1), hymenistatin 1, and hymenamides G, H, and J, were isolated from the South China Sea sponge Phakellia fusca. Their structures were elucidate

Full text of DOI:10.1021/np9008267

650
J. Nat. Prod. 2010, 73, 650–655
Proline-Containing Cyclopeptides from the Marine Sponge Phakellia fusca
Hong-Jun Zhang,†,‡,§ Yang-Hua Yi,‡ Gen-Jin Yang,‡ Min-Ye Hu,† Gui-Dong Cao,† Fan Yang,† and Hou-Wen Lin*,†
Laboratory of Marine Drugs, Department of Pharmacy, Changzheng Hospital, Second Military Medical UniVersity, 415 Fengyang Road,
Shanghai 200003, People’s Republic of China, Research Center for Marine Drugs, School of Pharmacy, Second Military Medical UniVersity,
325 Guohe Road, Shanghai 200433, People’s Republic of China, and Hospital of PLA Troops 95746, Qionglai 611531, People’s Republic of
China
ReceiVed December 18, 2009
Four new cyclopeptides, phakellistatins 15-18 (2-5), together with five known cyclopeptides, phakellistatin 13 (1),
hymenistatin 1, and hymenamides G, H, and J, were isolated from the South China Sea sponge Phakellia fusca. Their
structures were elucidated by HR-ESIMS, NMR, and MALDI-TOF/TOF sequence analysis. The absolute configurations
of the amino acid residues of 2-5 were assigned to be L by enantioselective HPLC analysis.
Marine organisms are prolific producers of structurally novel
natural products with significant biological activities, which have
been considered as promising resources for lead compounds or drug
candidates.1 Marine sponges of the genus Phakellia (order Hali-
chondrida, family Axinellidae) have attracted a great deal of
attention for having bioactive cyclopeptides (phakellistatins),2
alkaloids,3 and polyethers.4 Besides phakellistatins, some structur-
ally similar cyclopeptides have been obtained from the sponges of
the order Halichondrida, such as axinastatins5 and axinellins6 (from
the genus Axinella), hymenistatin 17 and hymenamides8 (from the
genus Hymeniacidon), stylopeptides,9 stylostatin,10 and wainunua-
mide11 (from the genus Stylotella), and stylisins12 and stylissa-
mides13 (from the genus Stylissa). These cyclopeptides commonly
consist of seven to 10 amino acid residues including at least one
proline residue, and some of them showed cancer cell line
cytotoxicity and antifungal activity. Studies by Pettit’s group showed
that phakellistatins were trace secondary metabolites of sponges
of the genus Phakellia.2 Our previous studies on the chemical
constituents of the marine sponge Phakellia fusca (500 g, dry wt)
collected from the South China Sea led to the isolation of a cytotoxic
cyclopeptide, phakellistatin 13 (1).2b In our reinvestigation aimed
at bioactive cyclopeptides of P. fusca on a larger scale (15 kg, dry
wt), four new proline-containing cyclopeptides, phakellistatins
15-18 (2-5), together with five known cyclopeptides, phakellistatin
13 (1), hymenistatin 1, and hymenamides G, H, and J, were obtained
from the CH2Cl2-soluble and n-BuOH-soluble extracts. Herein, we
report the isolation and structure elucidation of these proline-
containing cyclopeptides (2-5) along with the in Vitro cytotoxicity
of phakellistatins 15 (2) and 16 (3).
Results and Discussion
The EtOH extract of dried P. fusca was partitioned between
EtOAc and H2O. The EtOAc phase extract was subjected to solvent
Phakellistatin 15 (2) was obtained as a glassy amorphous solid
from MeOH, and its molecular formula was established as
C48H71N9O9 from the positive ion HR-TOF-ESIMS peak at m/z
940.5276 and the 13C NMR data. The 1H and 13C NMR spectra of
2 in DMSO-d6 were characteristic of a peptide.14 The 13C NMR
spectrum exhibited eight amide carbonyl carbons, as well as eight
partitioning to yield a CH2Cl2-soluble extract, and the H2O phase
fraction was extracted by n-BuOH to give an n-BuOH-soluble
extract. By column chromatography or vacuum liquid chromatog-
raphy (on Sephadex LH-20, ODS silica, and silica gel) and RP-
HPLC, three new cyclopeptides, 2, 4, and 5, together with four
known cyclopeptides (1, hymenistatin 1, and hymenamides G and
H) were obtained from the CH2Cl2-soluble extract. Similarly, one
new cyclopeptide (3) and the known hymenamide J were isolated
from the n-BuOH-soluble extract.
1
R-amino acid carbons (Table 1). The H NMR spectrum showed
five amide NH signals and one hydroxy proton signal at δH 5.22
(1H, d, J ) 12.8 Hz). Eight aromatic carbons and a NH signal at
δH 10.76 (br s) suggested the existence of a Trp residue (Table 1).
Detailed analysis of the HMQC, COSY, HMBC, TOCSY, and
HMQC-TOCSY spectra allowed the identification of eight amino
acid residues as Pro (3×), Leu (2×), Ile, Trp, and Thr. According
to the restrictions of the molecular formula and the corresponding
* To whom correspondence should be addressed. Tel/Fax: +86-21-
65585154. E-mail: franklin67@126.com.
† Changzheng Hospital, Second Military Medical University.
‡ School of Pharmacy, Second Military Medical University.
§ Hospital of PLA Troops 95746.
10.1021/np9008267  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/26/2010

Cyclopeptides from Phakellia fusca
Journal of Natural Products, 2010, Vol. 73, No. 4 651
Table 1. 1H (600 MHz) and 13C NMR (150 MHz) Data for 2 in DMSO-d6
position
δC, mult.
δH (J in Hz)
position
δC, mult.
δH (J in Hz)
a: 2.00, m
b: 1.85, m
a: 3.99, m
b: 3.59, dt (7.0, 9.4)
Pro1
CO
R
γ
24.44, CH2
171.13, C
60.51, CH
28.71, CH2
3.85, t (7.2)
δ
47.91, CH2
ꢀ
a: 1.81, m
b: 1.63, m
a: 1.99, m
b: 1.81, m
a: 3.75, m
b: 3.46, m
Leu1
NH
CO
R
γ
δ
24.38, CH2
46.66, CH2
8.76, d (6.8)
169.74, C
52.96, CH
35.82, CH2
3.46, m
ꢀ
a: 2.16, m
b: 1.66, m
1.47, m
0.87, d (6.0)
0.85, d (7.3)
Trp
NH
CO
R
7.91, d (7.1)
γ
δ
δ′
Thr
NH
CO
R
ꢀ
γ
24.6, CH
20.92, CH3
23.28, CH3
170.30, C
55.66, CH
24.58, CH2
3.94, m
a: 3.47, m
b: 3.36, dd (14.4, 3.8)
ꢀ
7.49, d (9.4)
1
2
3
4
5
6
7
8
110.96, C
123.38, CH
168.77, C
56.12, CH
67.65, CH
18.99, CH3
6.97, br s
10.76, br s
4.87, dd (9.5, 3.5)
4.13, m
1.03, d (6.3)
5.22, d (12.8)
136.05, C
111.3, CH
120.76, CH
118.1, CH
117.93, CH
127.06, C
7.32, d (8.0)
7.05, t (8.0)
6.97, t (8.0)
7.46, d (8.0)
OH
Pro3
CO
R
170.93, C
59.07, CH
28.31, CH2
4.46, dd (8.7, 4.4)
a: 2.06, m
b: 1.92, m
a: 1.83, m
b: 1.72, m
9
ꢀ
Ile
NH
CO
R
ꢀ
7.40, d (9.8)
γ
δ
24.54, CH2
46.92, CH2
170.11, C
54.19, CH
33.01, CH
23.31, CH2
4.67, t (10.5)
2.02, m
a: 1.36, m
b: 1.03, m
0.76, t (7.4)
0.78, d (6.6)
a: 3.68, dt (9.8, 6.8)
b: 3.54, dt (9.8, 6.8)
γ
Leu2
NH
CO
R
8.13, d (8.6)
δ
9.52, CH3
15.07, CH3
170.87, C
48.14, CH
38.02, CH2
ꢀ-Me
Pro2
CO
R
4.44, m
ꢀ
a: 1.70, m
b: 1.05, m
1.54, m
0.81, d (6.6)
0.86, d (6.6)
170.55, C
60.32, CH
29.61, CH2
4.10, t (7.9)
a: 2.15, m
b: 1.71, m
γ
δ
δ′
24.36, CH
20.86, CH3
23.02, CH3
ꢀ
degrees of unsaturation, eight peptide bonds were essential for 2,
indicating 2 was a cyclopeptide.
13C NMR spectra of 3 exhibited two sets of eight amide NH, one
hydroxy proton, nine amide carbonyl carbons, and nine R-amino
acid carbons, which were indicative of a peptide (Table 2).14
Extensive inspection of the HMQC, COSY, HMBC, TOCSY, and
HMQC-TOCSY spectra allowed the identification of two sets of
nine amino acid residues as Pro, Phe, Asp, Ser, Arg, Ala, Val, Thr,
and Tyr, of which the Arg (m/z 100, 112, 129), Phe (m/z 120), and
Tyr (m/z 136) residues were indicated by their immonium and
related ions in the MALDI-TOF/TOF spectrum.18 For the major
conformer 3a, the HMBC correlations between Ser-NH/Asp-CO,
Ala-NH/Arg-CO, Val-NH/Ala-CO, and Tyr-NH/Thr-CO, together
with the ROESY correlations between Asp-HR/Ser-NH, Ser-NH/
Arg-NH, Arg-NH/Ala-NH, Ala-NH/Val-NH, Val-NH/Thr-NH, and
Thr-NH/Tyr-NH, exhibited the fragment of Asp-Ser-Arg-Ala-Val-
Thr-Tyr. The ROESY correlations between Tyr-HR/Pro-Hδ, Pro-
HR/Phe-NH, and Phe-HR/Asp-NH extended this fragment as
cyclo(Pro-Phe-Asp-Ser-Arg-Ala-Val-Thr-Tyr). The NMR spectra
of the minor conformer 3b exhibited similar HMBC and ROESY
correlations, showing the same peptide sequence as that of the major
conformer 3a (Figure 2). The MALDI-TOF/TOF sequence analysis
confirmed the connections of amino acid residues of 3 and showed
that the preferred ring-opening began at the Tyr-Pro peptide bond
(Figure 1).18a,19
Two fragments, Pro1-Trp-Ile and Pro2-Leu1-Thr-Pro3-Leu2, were
indicated by the HMBC correlations between Trp-NH/Pro1-CO, Ile-
NH/Trp-CO, Leu1-NH/Pro2-CO, Thr-NH/Leu1-CO, Pro3-Hδ/Thr-
CO, and Leu2-NH/Pro3-CO. The ROESY correlations between Ile-
HR/Pro2-Hδ and Leu2-HR/Pro1-Hδ allowed the establishment of
the sequence of 2 as cyclo(Pro1-Trp-Ile-Pro2-Leu1-Thr-Pro3-Leu2).
The ∆δCꢀ-Cγ values of the Pro residues (4.33, 5.17, and 3.77 ppm
for Pro1, Pro2, and Pro3, respectively) and the ROESY correlations
between Xaai-1-HR/Proi-Hδ suggested that all three Xaa-Pro amide
bonds were of the trans-configuration.13,15
The sequence of 2 was further confirmed by MALDI-TOF/TOF
sequence analysis. Although there was more than one possible ring-
opening position for the cyclopeptide, the preferred ring-opening
of 2 occurred at the Leu2-Pro1 amide bond due to the proline effect
on proline’s high proton affinity.16,17 The immonium and related
ions indicated the existence of Leu or Ile (m/z 86, 44), and Tyr
(m/z 136) residues.18 A linearized peptide 2, Pro1-Trp-Ile-Pro2-Leu1-
Thr-Pro3-Leu2, was demonstrated by a main series of adjacent
bn(+1) ions at m/z 805, 708, 607, 494, 397, and 284, corresponding
to the successive loss of Leu, Pro, Thr, Leu, Pro, Ile, and the
terminal dipeptide ion Pro-Trp (Figure 1).18a,19
Phakellistatin 16 (3) was obtained as a glassy, amorphous solid
from MeOH, and its molecular formula C48H68N12O14 was deduced
from the positive ion HR-TOF-ESIMS peak at m/z 1059.4883 and
the negative ion HR-TOF-ESIMS peak at m/z 1035.4917, as well
as the 13C NMR data. Phakellistatin 16 (3) existed as a mixture of
two conformers in common NMR solvents (CD3OH, CD3CN,
C5D5N, DMSO-d6, etc.), and the two sets of NMR signals in the
ratio of 3:1 in DMSO-d6 were relatively well resolved. The 1H and
The ∆δCꢀ-Cγ value (4.53 ppm) of the Pro residue in the major
conformer 3a and its ROESY correlation between Tyr-HR/Pro-Hδ
indicated that the Tyr-Pro amide bond in 3a was the trans-
configuration, while the ∆δCꢀ-Cγ value (8.77 ppm) of the Pro residue
in the minor conformer 3b and its ROESY correlation between
Tyr-HR/Pro-HR suggested that the Tyr-Pro amide bond in 3b was
the cis-configuration (Table 2 and Figure 2).13,15

652 Journal of Natural Products, 2010, Vol. 73, No. 4
Zhang et al.
Table 2. 1H (600 MHz) and 13C NMR (150 MHz) Data for 3 in
DMSO-d6
3a (major conformer)
δC, mult. δH (J in Hz)
3b (minor conformer)
position
δC, mult.
δH (J in Hz)
Pro
CO
R
171.23, C
60.64, CH
28.74, CH2 a: 1.77, m
b: 1.26, m
170.67, C
59.83, CH
29.88, CH2 a: 1.56, m
b: 0.95, m
4.00, m
3.51, m
ꢀ
γ
δ
24.21, CH2 a: 1.66, m
b:1.53, m
47.13, CH2 a: 3.63, m
b: 3.19, m
21.11, CH2 a: 1.57, m
b:1.41, m
45.74, CH2 a: 3.18, m
b: 3.01, m
Phe
NH
CO
R
7.23, br s
169.24, C
8.50, br s
170.05, C
54.44, CH
4.22, m
56.01, CH
4.22, m
ꢀ
34.87, CH2 a: 3.33, m
b: 2.90, m
35.54, CH2 a: 3.02, m
b: 2.96, m
1
138.56, C
138.11, C
2, 6
3, 5
4
128.88, CH 7.08, d (7.5)
128.10, CH 7.23, t (7.5)
126.19, CH 7.16, t (7.5)
128.88, CH 7.21, d (7.5)
128.03, CH 7.23, t (7.5)
126.19, CH 7.16, t (7.5)
Asp
NH
CO
R
7.00, d (8.8)
172.72, C
7.24, ov
172.72, C
50.19, CH
50.28, CH
4.71, br t (10.2)
4.34, br t (7.5)
40.03, CH2 a: 2.69, m
b: 2.55, m
ꢀ
41.96, CH2 a: 2.82, m
b: 2.17, m
ꢀ-CO
Ser
NH
CO
R
175.78, C
176.12, C
8.53, d (4.4)
169.89, C
57.36, CH
60.63, CH2 a: 3.78, m
b: 3.68, br d (7.8)
8.44, d (4.4)
170.28, C
57.24, CH
60.53, CH2 a: 3.76, m
b: 3.67, m
4.01, m
4.14, m
ꢀ
Figure 1. MALDI-TOF/TOF sequence ions (m/z) for protonated
molecular [M + H]+ ions of phakellistatins 15-18 (2-5).
OH
Arg
NH
CO
R
4.88, d (6.0)
8.76, d (7.2)
4.04, m
4.89, d (6.0)
8.26, d (6.4)
172.42, C
4.12, m
27.60, CH2 a: 1.99, m
b: 1.80, m
presence of eight amino acid residues as Pro (3×), Trp, Val, Leu,
171.49, C
53.33, CH
and Ile (2×) (Table 3).
52.79, CH
The HMBC correlations between Trp-NH/Pro1-CO and Leu-NH/
Pro2-CO and the ROESY correlations between Trp-HR/Val-NH and
Val-HR/Pro2-Hδ suggested the fragment of Pro1-Trp-Val-Pro2-Leu.
The ROESY correlations between Ile1-HR/Pro3-Hδ and Pro3-HR/
Ile2-NH indicated the existence of Ile1-Pro3-Ile2. The ROESY
correlations between Leu-HR/Ile1-HR and Ile2-HR/Pro1-Hδ con-
nected these two fragments, and accordingly the peptide 4 was
established as cyclo(Pro1-Trp-Val-Pro2-Leu-Ile1-Pro3-Ile2). This
sequence was further supported by the MALDI-TOF/TOF sequence
analysis, and the preferred ring-opening occurred at the Ile2-Pro1
amide bond (Figure 1).18a,19 The ∆δCꢀ-Cγ values of Pro residues
(4.10, 4.70, and 4.02 ppm for Pro1, Pro2, and Pro3, respectively)
and the ROESY correlations between Xaai-1-HR/Proi-Hδ indicated
thatallthreeXaa-Proamidebondswereofthetrans-configuration.13,15
Phakellistatin 18 (5) was obtained as a glassy, amorphous solid
from MeOH, and its molecular formula C45H61N7O8 was established
from the positive ion HR-TOF-ESIMS peak at m/z 850.4481 and
the 13C NMR data. Phakellistatin 18 (5) existed mainly as one
conformer in CD3OH. Examination of the HMQC, COSY, HMBC,
TOCSY, and HMQC-TOCSY spectra allowed the identification of
seven amino acid residues as Pro (3×), Ile (2×), Phe, and Tyr
(Table 4). The HMBC correlations between Tyr-NH/Pro1-CO, Phe-
NH/Ile1-CO, and Ile2-NH/Pro3-CO exhibited the fragments Pro1-
Tyr, Ile1-Phe, and Pro3-Ile2, respectively. There were no further
adequate HMBC or ROESY correlations for the complete sequence
assignment, which was finished by MALDI-TOF/TOF sequence
analysis. A linearized peptide 5, Pro1-Tyr-Pro2-Ile1-Phe-Pro3-Ile2,
was indicated by a series of adjacent bn(+1) ions, which suggested
that peptide 5 was cyclo(Pro1-Tyr-Pro2-Ile1-Phe-Pro3-Ile2), and the
preferred ring-opening occurred at the Ile2-Pro1 amide bond (Figure
1).18a,19 The ∆δCꢀ-Cγ values of the three Pro residues (9.58, 9.24,
and 8.99 ppm for Pro1, Pro2, and Pro3, respectively) showed that
all three Xaa-Pro amide bonds were of the cis-configuration.13,15
The absolute configurations of the amino acid residues of
phakellistatins 15-18 (2-5) were determined by enantioselective
ꢀ
27.80, CH2 a: 1.79, m
b: 1.72, m
γ
23.44, CH2 a: 1.58, m
b: 1.55, m
40.57, CH2 a: 3.14, m
b: 2.94, m
23.72, CH2 a: 1.58, m
b: 1.55, m
40.83, CH2 a: 3.14, m
b: 2.94, m
δ
δ-NH
guanidine 157.42, C
Ala
NH
CO
R
ꢀ
9.36, br s
9.36, br s
157.24, C
7.58, d (7.0)
7.93, d (4.8)
172.58, C
172.25, C
47.58, CH
4.44, qui (7.0)
49.94, CH
3.93, qui (6.5)
18.87, CH3 1.28, d (7.0)
16.75, CH3 1.29, d (7.0)
Val
NH
CO
R
8.08, d (6.3)
171.31, C
7.63, d (7.5)
170.94, C
59.96, CH
29.38, CH
3.98, t (7.0)
2.07, m
60.53, CH
28.96, CH
3.87, t (7.1)
2.16, m
ꢀ
γ
18.38, CH3 0.87, d (6.8)
19.43, CH3 0.89, d (6.8)
18.57, CH3 0.87, d (6.8)
19.46, CH3 0.91, d (6.8)
γ′
Thr
NH
CO
R
7.63, d (7.5)
169.67, C
7.49, d (7.5)
170.14, C
58.83, CH
65.58, CH
3.98, m
4.15, m
5.03, br s
57.53, CH
65.70, CH
4.27, m
4.11, m
5.88, br s
ꢀ
OH
γ
20.63, CH3 0.98, d (6.4)
20.54, CH3 0.99, d (6.4)
Tyr
NH
CO
R
7.01, d (8.2)
170.41, C
7.83, d (8.2)
169.89, C
51.15, CH
4.81, q (7.4)
53.33, CH
4.59, br s
ꢀ
36.57, CH2 a: 2.83, m
b: 2.94, m
126.92, C
130.06, CH 7.06, d (8.3)
115.11, CH 6.64, d (8.3)
155.90, C
36.94, CH2 a: 3.04, m
b: 2.60, m
126.19, C
130.41, CH 6.92, d (8.3)
115.11, CH 6.62, d (8.3)
156.18, C
1
2, 6
3, 5
4
Phakellistatin 17 (4) was obtained as a glassy, amorphous solid
from MeOH, and its molecular formula was determined to be
C49H73N9O8 from the positive ion HR-TOF-ESIMS peak at m/z
938.5482 and the 13C NMR data. Phakellistatin 17 (4) appeared
essentially as one conformer in DMSO-d6. Inspection of the HMQC,
COSY, HMBC, TOCSY, and HMQC-TOCSY spectra revealed the

Cyclopeptides from Phakellia fusca
Journal of Natural Products, 2010, Vol. 73, No. 4 653
Figure 2. Key HMBC and ROESY correlations for phakellistatin 16 (3).
Table 3. 1H (600 MHz) and 13C NMR (150 MHz) Data for 4 in DMSO-d6
position
δC, mult.
δH (J in Hz)
position
δC, mult.
δH (J in Hz)
Pro1
CO
R
δ
47.29, CH2
a: 3.87, m
b: 3.59, m
170.90, C
60.92, CH
28.83, CH2
3.88, m
Leu
NH
CO
R
ꢀ
a: 1.94, m
b: 1.78, m
a: 1.76, m
b: 1.73, m
a: 3.59, m
b: 3.49, m
8.25, d (6.8)
170.11, C
54.09, CH
37.29, CH2
γ
δ
24.73, CH2
47.05, CH2
4.50, m
ꢀ
a: 2.14, m
b: 1.60, m
1.47, m
0.85, d (6.8)
0.87, d (6.8)
γ
δ
δ′
Ile1
NH
CO
R
24.46, CH
20.65, CH3
23.23, CH3
Trp
NH
CO
R
8.20, d (6.8)
169.75, C
56.46, CH
23.51, CH2
3.75, m
a: 3.62, m
b: 3.40, dd (14.5, 3.1)
7.55, d (8.0)
ꢀ
171.50, C
53.93, CH
37.96, CH
23.16, CH2
4.65, m
1.74, m
a: 1.31, m
b: 1.15, m
0.75, t (7.6)
0.85, d (6.6)
1
2
3
4
5
6
7
8
111.43, C
123.49, CH
ꢀ
γ
6.98, br s
10.73, br s
136.11, C
δ
10.94, CH3
14.83, CH3
111.23, CH
120.71, CH
117.98, CH
118.07, CH
127.01, C
7.32, d (8.0)
7.05, t (8.0)
6.96, t (8.0)
7.47, d (8.0)
ꢀ-Me
Pro3
CO
R
169.85, C
59.41, CH
28.83, CH2
4.54, m
9
ꢀ
a: 2.12, m
b: 1.71, m
a: 1.83, m
b: 1.73, m
a: 3.50, m
b: 3.44, m
Val
NH
CO
R
ꢀ
γ
7.71, d (9.0)
γ
δ
24.81, CH2
47.35, CH2
169.36, C
55.81, CH
29.85, CH
18.99, CH3
19.46, CH3
4.52, t (10.0)
2.11, m
0.78, d (6.9)
0.89, d (6.9)
Ile2
NH
CO
R
ꢀ
γ
γ′
Pro2
CO
R
8.32, br d (6.0)
169.56, C
54.18, CH
38.40, CH
24.23, CH2
172.01, C
60.52, CH
29.35, CH2
4.39, t (9.1)
1.48, m
a: 1.32, m
b: 0.98, m
0.76, t (7.3)
0.88, d (8.0)
4.13, t (7.3)
a: 2.09, m
b: 1.73, m
a: 2.01, m
b: 1.86, m
ꢀ
γ
24.65, CH2
δ
ꢀ-Me
10.28, CH3
14.14, CH3
HPLC analysis after hydrolysis of 2-5. All amino acids were found
to possess the L-configurations.
matter of fact, cyclopeptides from the genus Phakellia were obtained
using similar isolation schemes, and most of them were isolated
from the CH2Cl2-soluble extract.2 Phakellistatin 16 (3), which has
a divergent backbone compared with other cyclopeptides from the
sponges of the order Halichondrida, is the first example from the
n-BuOH-soluble extract of the genus Phakellia, and its hydrophi-
licity can be ascribed to the hydrophilic residues Arg, Asp, Ser,
and Thr. Phakellistatin 15 (2) appears as one conformer in DMSO-
d6, while phakellistatins 16-18 (3-5) exist as more than one
conformer in common solvents. The proline residue of phakellistatin
16 (3) bears trans- and cis-configurations in the major and the minor
Phakellistatins 15-18 (2-5) are new proline-containing cyclo-
peptides from the South China Sea sponge P. fusca. Phakellistatins
15 (2) and 17 (4), with analogous sequences Pro-Trp-Val/Ile-Leu-
Thr/Ile-Pro-Leu/Ile, are structurally similar to hymenamide H, which
was originally from the Okinawan sponge Hymeniacidon sp.,8b
supporting the remarkable analogy in the cyclopeptides from the
genera Axinella, Hymeniacidon, Phakellia, Stylotella, and Stylissa.6b,13
Phakellistatin 18 (5) has the same seven residues as those of
phakellistatins 12j and 22i and only differs in the sequence. As a

654 Journal of Natural Products, 2010, Vol. 73, No. 4
Zhang et al.
Table 4. 1H (600 MHz) and 13C NMR (150 MHz) Data for 5 in
CD3OH
Company); 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 Chirex 3126 (D)-penicillamine column
(Phenomenex, 150 × 4.6 mm). The commercial amino acids used for
enantioselective analysis were from Sigma-Aldrich Chemical Corporation.
Animal Material. Specimens of Phakellia fusca were collected
around Yongxing Island and seven connected islets in the South China
Sea during June 2007 and were identified by Prof. Jin-He Li (Institute
of Oceanology, Chinese Academy of Sciences, China). A voucher
sample (No. DS-PF02) was deposited in the Laboratory of Marine
Drugs, Department of Pharmacy, Changzheng Hospital, Second Military
Medical University, China. The sponge P. fusca is of great abundance
in the Xisha Islands, and therefore this collection was considered to
have no significant adverse ecological effect.
Extraction and Purification. The sponge (15 kg, dry wt) was
extracted with 95% EtOH, and combined EtOH extracts were concen-
trated under reduced pressure. This extract was suspended in H2O and
extracted in turn with EtOAc and n-BuOH to afford the EtOAc-soluble
extract and the n-BuOH-soluble extract. The EtOAc-soluble extract was
partitioned between MeOH-H2O (9:1) and petroleum ether to yield a
brownish-red oil (358 g). The MeOH-H2O (9:1) phase was diluted to
3:2 with water and extracted with CH2Cl2 to afford the CH2Cl2-soluble
extract (55 g). This CH2Cl2-soluble extract was subjected to VLC on
silica gel using CH2Cl2-MeOH (50:1, 20:1, 15:1, 10:1, 5:1, and 2:1)
as eluent to give 10 subfractions (A-J). Subfraction D was subjected
to CC on Sephadex LH-20 and ODS silica and further purified by HPLC
(YMC-Pack Pro C18 RS, 5 µm, 10 × 250 mm, 1.5 mL/min, UV
detection at 215 and 280 nm) eluting with CH3OH-H2O (90:10) to
yield pure peptides 5 (11.5 mg) and 1 (120 mg). Similarly, new peptides
2 (10.8 mg) and 4 (19.4 mg), together with three known peptides,
hymenistatin 1(16.8 mg), hymenamide G (18.0 mg), and hymenamide
H (21.5 mg), were purified from subfraction E. The n-BuOH-soluble
extract was subjected to CC on Sephadex LH-20 to afford three
subfractions (K-M). Subfraction K was subjected to CC on ODS silica
and further purified by HPLC to yield pure peptides 3 (33.8 mg) and
hymenamide J (35.2 mg).
position δC, mult. δH (J in Hz) position δC, mult. δH (J in Hz)
Pro1
CO
R
γ
26.75, CH2 a: 1.43, m
b: 1.14, m
10.72, CH3 0.83, t (7.5)
16.11, CH3 0.78, d (6.8)
172.23, C
62.46, CH 3.51, br d (8.4)
32.08, CH2 a: 1.72, m
b: 1.30, m
22.50, CH2 a: 1.58, m
b: 1.41, m
δ
ꢀ
ꢀ-Me
Phe
NH
CO
R
γ
δ
8.30, d (7.8)
173.75, C
53.31, CH 4.61, m
39.60, CH2 a: 2.97, m
b: 2.91, m
47.21, CH2 a: 3.43, m
b: 3.12, m
ꢀ
Tyr
NH
CO
R
8.67, d (8.2)
170.44, C
54.27, CH 4.57, m
37.37, CH2 a: 3.03, m
b: 2.98, m
1
137.42, C
2, 6
3, 5
4
130.26, CH 7.18, d (7.5)
129.74, CH 7.30, t (7.5)
128.20, CH 7.25, t (7.5)
ꢀ
Pro3
CO
R
1
127.27, C
172.53, C
2, 6
3, 5
4
131.71, CH 6.78, d (8.4)
115.99, CH 6.62, d (8.4)
157.60, C
59.58, CH 4.48, br d (7.8)
31.49, CH2 a: 2.06, m
b: 1.91, m
22.50, CH2 a: 2.21, m
b: 1.83, m
ꢀ
Pro2
CO
R
γ
δ
173.32, C
62.72, CH 4.43, br d (8.1)
32.13, CH2 a: 2.41, m
b: 2.03, m
22.89, CH2 a: 1.93, m
b: 1.64, m
48.04, CH2 a: 3.56, m
b: 3.36, m
ꢀ
Ile2
NH
CO
R
ꢀ
γ
γ
δ
8.39, br s
172.72, C
58.67, CH 4.00, br d (7.8)
37.49, CH 1.77, m
26.44, CH2 a: 1.72, m
b: 1.33, m
47.78, CH2 a: 3.55, m
b: 3.44, m
Ile1
NH
CO
R
9.33, br s
172.18, C
59.91, CH 3.93, t (9.0)
35.89, CH 1.90, m
δ
ꢀ-Me
11.19, CH3 0.83, t (7.8)
15.28, CH3 0.96, d (7.1)
ꢀ
conformers, respectively, implying that the proline residue greatly
affects the solvent conformations. Although conformers in solution
may lead to overlapping signals in the NMR spectra disadvanta-
geous to the structure determination, the presence of the proline
residue in phakellistatins 16-18 (3-5) can facilitate the TOF/TOF
sequence analysis.16,17
The new cyclopeptides 2-5 were tested for cytotoxic activity
in Vitro. Phakellistatin 15 (2) exhibited cytotoxicity against cancer
cell line P388 with an IC50 value of 8.5 µM. Phakellistatin 16 (3)
showed cytotoxicity against cancer cell lines P388 and BEL-7402
with IC50 values of 5.4 and 14.3 µM, respectively. Phakellistatins
17 (4) and 18 (5) showed no cytotoxicity against the cancer cell
lines P388 and BEL-7402 in this assay.
Phakellistatin 15 (2, cyclo(L-Pro-L-Trp-L-Ile-L-Pro-L-Leu-L-
Thr-L-Pro-L-Leu)): glassy, amorphous solid; mp 213-215 °C; [R]19
D
-167 (c 0.120, MeOH); NMR data, see Table 1; MALDI-TOF/TOF
data, see Figure 1; HR-TOF-ESIMS m/z 940.5276 [M + Na]+ (calcd
for C48H71N9O9Na, 940.5272).
Phakellistatin 16 (3, cyclo(L-Pro-L-Phe-L-Asp-L-Ser-L-Arg-L-
Ala-L-Val-L-Thr-L-Tyr)): glassy, amorphous solid; mp >300 °C dec;
[R]19 -38 (c 0.100, MeOH); NMR data, see Table 2; MALDI-TOF/
D
TOF data, see Figure 1; HR-TOF-ESIMS m/z 1059.4883 [M + Na]+
(calcd for C48H68N12O14Na, 1059.4876) and m/z 1035.4917 [M - H]-
(calcd for C48H67N12O14, 1035.4900).
Five known cyclopeptides, phakellistatin 13 (1),2b hymenistatin
1,7 and hymenamides G, H, and J,8b were determined on the basis
of high-resolution TOF-ESIMS, 1D- and 2D-NMR experiments
including HMQC, COSY, HMBC, TOCSY, HMQC-TOCSY, and
ROESY, and MALDI-TOF/TOF sequence analysis. Notably, hy-
menamide J existed as a mixture of two conformers in DMSO-d6,
affording complicated NMR spectra, but its sequence could be
established adequately by a MALDI-TOF/TOF experiment, whereas
the sequence was previously achieved by Edman degradation of
its partial hydrolysates.8b
Phakellistatin 17 (4, (cyclo(L-Pro-L-Trp-L-Val-L-Pro-L-Leu-L-
Ile-L-Pro-L-Ile)): glassy, amorphous solid; mp 194-196 °C; [R]19
D
-126 (c 0.055, MeOH); NMR data, see Table 3; MALDI-TOF/TOF
data, see Figure 1; HR-TOF-ESIMS m/z 938.5482 [M + Na]+ (calcd
for C49H73N9O8Na, 938.5480).
Phakellistatin 18 (5, cyclo(L-Pro-L-Tyr-L-Pro-L-Ile-L-Phe-L-
Pro-L-Ile)): glassy, amorphous solid; mp 194-196 °C; [R]19 -104
D
(c 0.280, MeOH); NMR data, see Table 4; MALDI-TOF/TOF data,
see Figure 1; HR-TOF-ESIMS m/z 850.4481 [M + Na]+ (calcd for
C45H61N7O8, 850.4479).
Hydrolyses of Phakellistatins 15-18 (2-5). Each of the peptides
2-5 (1.0 mg) was dissolved in 6 N HCl (1 mL) in a sealed tube and
heated at 110 °C for 24 h. After cooling, the liquid was evaporated
under N2, and then the residue was dried under vacuum to yield the
hydrolysates.
Phakellistatins 15-18 (2-5): Configurational Assignments. The
hydrolysates of peptides 2-5 and authentic L- and D-amino acids were
analyzed using a ligand-exchange type Chirex 3126 (D)-penicillamine
column (150 × 4.6 mm, N,S-dioctyl-(D)-penicillamine complexed with
Cu2+) with aqueous CuSO4 or CuSO4-MeOH as mobile phase on a
Waters 1525/2998 HPLC instrument. All amino acid residues in
phakellistatins 15-18 (2-5) were revealed 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-MeOH (85:
15), flow rate at 1 mL/min, L-Ile (15.7), D-Ile (24.4), L-Leu (17.2),
D-Leu (25.7), L-Tyr (18.0), D-Tyr (26.4); (2) aqueous 2 mM
Experimental Section
General Experimental Procedures. Optical rotation data were
recorded on a JASCO P-1030 polarimeter. Melting points were
measured on a SCW X-4 melting point apparatus and were uncorrected.
The NMR experiments were conducted with a Bruker AVANCE-600
instrument. High-resolution TOF-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, USA). Reversed-phase HPLC was performed on a YMC-
Pack Pro C18 RS column (250 × 10 mm, 5 µm) using a Waters 1525
HPLC instrument with a Waters 2998 UV detector. Column chroma-
tography (CC) was performed on Sephadex LH-20 (Pharmacia) and
YMC ODS-A (50 µm). Vacuum liquid chromatography (VLC) was
performed on silica gel (200-300 mesh, Qingdao Ocean Chemical

Cyclopeptides from Phakellia fusca
Journal of Natural Products, 2010, Vol. 73, No. 4 655
Herald, D. L.; Williams, M. D.; Doubek, D. L.; Schmidt, J. M.; Tackett,
L. P.; Brune, D. C. J. Nat. Prod. 1993, 56, 260–267. (k) Pettit, G. R.;
Tan, R. Bioorg. Med. Chem. Lett. 2003, 13, 685–688.
CuSO4-MeOH (70:30), flow rate at 1 mL/min, L-Phe (17.4), D-Phe
(23.5), L-Trp (48.8), D-Trp (52.9); (3) aqueous 1 mM CuSO4, flow rate
at 1 mL/min, L-Ala (6.0), D-Ala (8.4), L-Arg (4.0), D-Arg (6.1), L-Pro
(13.1), D-Pro (30.9), L-Val (19.7), D-Val (34.4); (4) aqueous 1 mM
CuSO4, flow rate at 0.5 mL/min, L-Asp (9.2), D-Asp (9.7); (5) aqueous
1 mM CuSO4, flow rate at 0.2 mL/min, L-Ser (30.2), D-Ser (33.5); (6)
aqueous 0.5 mM CuSO4, flow rate at 0.5 mL/min, L-Thr (15.6), D-Thr
(18.5).
(3) Pettit, G. R.; McNulty, J.; Herald, D. L.; Doubek, D. L.; Chapuis,
J.-C.; Schmidt, J. M.; Tackett, L. P.; Boyd, M. R. J. Nat. Prod. 1997,
60, 180–183.
(4) (a) Sakai, R.; Rinehart, K. L. J. Nat. Prod. 1995, 58, 773–777. (b)
Pettit, G. R.; Ichihara, Y.; Wurzel, G.; Williams, M. D.; Schmidt, J. M.;
Chapuis, J.-C. J. Chem. Soc., Chem. Commun. 1995, 383–385. (c)
Pettit, G. R.; Tan, R.; Gao, F.; Williams, M. D.; Doubek, D. L.; Boyd,
M. R.; Schmidt, J. M.; Chapuis, J. C.; Hamel, E.; Bai, R.; Hooper,
J. N. A.; Tackett, L. P. J. Org. Chem. 1993, 58, 2538–2543.
(5) (a) Pettit, G. R.; Herald, C. L.; Boyd, M. R.; Leet, J. E.; Dufresne, C.;
Doubek, D. L.; Schmidt, J. M.; Cerny, R. L.; Hooper, J. N. A.; Rutzler,
K. C. J. Med. Chem. 2002, 34, 3339–3340. (b) Pettit, G. R.; Gao, F.;
Schmidt, J. M.; Chapuis, J.-C.; Cerny, R. L. Bioorg. Med. Chem. Lett.
1994, 4, 2935–2940. (c) Pettit, G. R.; Gao, F.; Cerny, R. L.; Doubek,
D. L.; Tackett, L. P.; Schmidt, J. M.; Chapuis, J.-C. J. Med. Chem.
1994, 37, 1165–1168. (d) Pettit, G. R.; Gao, F.; Cerny, R. Heterocycles
1993, 35, 711–718.
(6) (a) Tabudravu, J. N.; Morris, L. A.; den Bosch, J. J. K.; Jaspars, M.
Tetrahedron 2002, 58, 7863–7868. (b) Randazzo, A.; Piaz, F. D.; Orru`,
S.; Debitus, C.; Roussakis, C.; Pucci, P.; Gomez-Paloma, L. Eur. J.
Org. Chem. 1998, 2659–2665.
MALDI-TOF/TOF Sequence Analysis of Phakellistatins 15-18
(2-5) and Known Cyclopeptides. The peptides were dissolved in 0.5
µL of matrix solution (R-cyano-4-hydroxycinnamic acid (CHCA) in
0.1% TFA, 50% ACN) before being spotted on the target plate. Samples
were analyzed with a 4700 MALDI-TOF/TOF Proteomics analyzer
(Applied Biosystems, USA) after air-drying. The UV laser was operated
at a 200 Hz repetition rate with a wavelength of 355 nm, and the
accelerated voltage operated at 20 kV. Myoglobin digested by trypsin
was used to calibrate the mass instrument with internal calibration mode.
All acquired spectra of samples were processed using 4700 Explore
software (Applied Biosystems) in a default mode. Parent mass peaks
([M + H]+) were picked out for tandem TOF/TOF analysis.
Cytotoxicity Assay. Cytotoxicity was evaluated as IC50 values by
using the MTT assay as described previously.20 Compounds were
solubilized in DMSO, with the working concentration of test substances
ranged from 1 to 100 µg/mL. Cells were inoculated into 96-well plates.
After incubation for 24 h, the cells were treated with various
concentrations of test substances for 48 h and then were incubated with
1 mg/mL MTT at 37 °C for 4 h, followed by solubilization in DMSO.
The formazan dye product was measured by the absorbance at 570 nm
on a microplate reader.
(7) Pettit, G. R.; Clewlow, P. J.; Dufresne, C.; Doubek, D. L.; Cerny,
R. L.; Rutzler, K. Can. J. Chem. 1990, 68, 708–711.
(8) (a) Kobayashi, J. i.; Nakamura, T.; Tsuda, M. Tetrahedron 1996, 52,
6355–6360. (b) Tsuda, M.; Sasaki, T.; Kobayashi, J. i. Tetrahedron
1994, 50, 4667–4680. (c) Tsuda, M.; Shigemori, H.; Mikami, Y.;
Kobayashi, J. i. Tetrahedron 1993, 49, 6785–6796. (d) Kobayashi, J.
i.; Tsuda, M.; Nakamura, T.; Mikami, Y.; Shigemori, H. Tetrahedron
1993, 49, 2391–2402.
(9) (a) Brennan, M. R.; Costello, C. E.; Maleknia, S. D.; Pettit, G. R.;
Erickson, K. L. J. Nat. Prod. 2008, 71, 453–456. (b) Pettit, G. R.;
Srirangam, J. K.; Herald, D. L.; Xu, J.-p.; Boyd, M. R.; Cichacz, Z.;
Kamano, Y.; Schmidt, J. M.; Erickson, K. L. J. Org. Chem. 1995, 60,
8257–8261.
(10) (a) Pettit, G. R.; Srirangam, J. K.; Herald, D. L.; Erickson, K. L.;
Doubek, D. L.; Schmidt, J. M.; Tackett, L. P.; Bakus, G. J. J. Org.
Chem. 1992, 57, 7217–7220. (b) Pettit, G. R.; Srirangam, J. K.; Herald,
D. L.; Erickson, K. L.; Doubek, D. L.; Schmidt, J. M.; Tackett, L. P.;
Bakus, G. J. J. Org. Chem. 1993, 58, 3222.
(11) Tabudravu, J.; Morris, L. A.; Kettenes-van den Bosch, J. J.; Jaspars,
M. Tetrahedron Lett. 2001, 42, 9273–9276.
(12) Mohammed, R.; Peng, J.; Kelly, M.; Hamann, M. T. J. Nat. Prod.
2006, 69, 1739–1744.
(13) Schmidt, G.; Grube, A.; Ko¨ck, M. Eur. J. Org. Chem. 2007, 4103–
4110.
Acknowledgment. This work was financially supported by the
National High Technology Research and Development Program of
China (863 Project, No. 2006AA09Z423) and the National Natural
Science Foundation of China (No. 20772154), and partially supported
by the National S & T Major Project of China (No. 2009ZX09103-
427) and the Major Program of Modernization of Chinese Medicine
(STCSM, 09dZ1975800).
Supporting Information Available: MALDI-TOF/FOF and NMR
spectra of new peptides 2-5, and NMR data tables and MALDI-TOF/
FOF data for known peptides 1, hymenistatin 1, and hymenamides G,
H, and J. This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) Bross-Walch, N.; Ku¨hn, T.; Moskau, D.; Zerbe, O. Chem. BiodiVersity
References and Notes
2005, 2, 147–177.
(1) (a) Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P. Nat.
ReV. Drug DiscoVery 2009, 8, 69–85. (b) Blunt, J. W.; Copp, B. R.;
Hu, W. P.; Munro, M. H.; Northcote, P. T.; Prinsep, M. R. Nat. Prod.
Rep. 2009, 26, 170–244. (c) Newman, D. J.; Cragg, G. M. Curr. Med.
Chem. 2004, 11, 1693–1713. (d) Newman, D. J.; Cragg, G. M. J. Nat.
Prod. 2004, 67, 1216–1238.
(15) Siemion, I. Z.; Wieland, T.; Pook, K.-H. Angew. Chem., Int. Ed. Engl.
1975, 14, 702–703.
(16) (a) Tomer, K. B.; Crow, F. W.; Gross, M. L.; Kopple, K. D. Anal.
Chem. 1984, 56, 880–886. (b) Hunt, D. F.; Yates, J. R.; Shabanowitz,
J.; Winston, S.; Hauer, C. Proc. Natl. Acad. Sci. U. S. A. 1986, 83,
6233–6237. (c) Schwartz, B. L.; Bursey, M. M. Biol. Mass Spectrom.
1992, 21, 92–96.
(17) Although the proline effect was mostly explained by the proline’s high
proton affinity (i.e., the greater basicity of the proline nitrogen), other
considerations have been proposed. (a) Vaisar, T.; Urban, J. J. Mass
Spectrom. 1996, 31, 1185–1187. (b) Breci, L. A.; Tabb, D. L.; Yates,
J. R.; Wysocki, V. H. Anal. Chem. 2003, 75, 1963–1971. (c) Paizs,
B.; Suhai, S. Mass Spectrom. ReV. 2005, 24, 508–548.
(2) (a) Pettit, G. R.; Tan, R. J. Nat. Prod. 2005, 68, 60–63. (b) Li, W.-L.;
Yi, Y.-H.; Wu, H.-M.; Xu, Q.-Z.; Tang, H.-F.; Zhou, D.-Z.; Lin, H.-
W.; Wang, Z.-H. J. Nat. Prod. 2003, 66, 146–148. (c) Pettit, G. R.;
Xu, J.-p.; Dorsaz, A.-C.; Williams, M. D. Bioorg. Med. Chem. Lett.
1995, 5, 1339–1344. (d) Pettit, G. R.; Xu, J.-p.; Cichacz, Z.; Schmidt,
J. M.; Dorsaz, A.-C.; Boyd, M. R.; Cerny, R. L. Heterocycles 1995,
40, 501–506. (e) Pettit, G. R.; Tan, R.; Ichihara, Y.; Williams, M. D.;
Doubek, D. L.; Tackett, L. P.; Schmidt, J. M. J. Nat. Prod. 1995, 58,
961–965. (f) Pettit, G. R.; Xu, J.-p.; Cichacz, Z. A.; Williams, M. D.;
Dorsaz, A.-C.; Brune, D. C.; Boyd, M. R.; Cerny, R. L. Bioorg. Med.
Chem. Lett. 1994, 4, 2091–2096. (g) Pettit, G. R.; Xu, J.-p.; Cichacz,
Z. A.; Williams, M. D.; Chapuis, J.-C.; Cerny, R. L. Bioorg. Med.
Chem. Lett. 1994, 4, 2677–2682. (h) Pettit, G. R.; Tan, R.; Herald,
D. L.; Williams, M. D.; Cerny, R. L. J. Org. Chem. 1994, 59, 1593–
1595. (i) Pettit, G. R.; Tan, R.; Williams, M. D.; Tackett, L.; Schmidt,
J. M.; Cerny, R. L.; Hooper, J. N. A. Bioorg. Med. Chem. Lett. 1993,
3, 2869–2874. (j) Pettit, G. R.; Cichacz, Z.; Barkoczy, J.; Dorsaz, A. C.;
(18) (a) Papayannopoulos, I. A. Mass Spectrom. ReV. 1995, 14, 49–73. (b)
Falick, A. M.; Hines, W. M.; Medzihradszky, K. F.; Baldwin, M. A.;
Gibson, B. W. J. Am. Soc. Mass. Spectrom. 1993, 4, 882–893.
(19) (a) Paizs, B.; Suhai, S. J. Am. Soc. Mass. Spectrom. 2004, 15, 103–
113. (b) Yergey, A. L.; Coorssen, J. R.; Backlund, P. S.; Blank, P. S.;
Humphrey, G. A.; Zimmerberg, J.; Campbell, J. M.; Vestal, M. L.
J. Am. Soc. Mass. Spectrom. 2002, 13, 784–791.
(20) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
NP9008267