Stylissamides e and F, cyclic heptapeptides from the caribbean sponge stylissa caribica

Article abstract of DOI:10.1021/np900664f

In addition to the four known stylissamides, A (1), B (2), C, and D, two new cyclic heptapeptides, stylissamides E (3) and F (4), were isolated from the Caribbean sponge Stylissa caribica. The structures of 3 and 4 were elucidated from a combination of mass spectrometric and NMR spectroscopic data as cyclo-(cis-Pro¹-Tyr²-trans-Pro³-Ala ⁴-Ile⁵-Gln⁶-Ile⁷) for stylissamide E (3) and cyclo-(trans-Pro¹-cis-Pro²-Phe ³-Asp⁴-trans-Pro⁵-Arg⁶-Phe ⁷) for stylissamide F (4).

Full text of DOI:10.1021/np900664f

7
38
J. Nat. Prod. 2010, 73, 738–742
Stylissamides E and F, Cyclic Heptapeptides from the Caribbean Sponge Stylissa caribica
Christine Cychon and Matthias K o¨ ck*
Alfred-Wegener-Institut f u¨ r Polar- und Meeresforschung in der Helmholtz-Gemeinschaft, Am Handelshafen 12, 27570 BremerhaVen, Germany
ReceiVed October 20, 2009
In addition to the four known stylissamides, A (1), B (2), C, and D, two new cyclic heptapeptides, stylissamides E (3)
and F (4), were isolated from the Caribbean sponge Stylissa caribica. The structures of 3 and 4 were elucidated from
1
2
3
4
5
6
a combination of mass spectrometric and NMR spectroscopic data as cyclo-(cis-Pro -Tyr -trans-Pro -Ala -Ile -Gln -
7
1
2
3
4
5
6
7
Ile ) for stylissamide E (3) and cyclo-(trans-Pro -cis-Pro -Phe -Asp -trans-Pro -Arg -Phe ) for stylissamide F (4).
Marine sponges are known to be a rich source of non-ribosomal
cyclic peptides with seven to ten amino acids that show various
biological activities. The cyclic nature of these metabolites often
n-BuOH phase was purified by size exclusion chromatography with
Sephadex LH-20.
1
Using HPLC-MS screening four proline-rich cyclic heptapep-
tides, stylissamides A (1), B (2), C, and D, were detected. In
addition, this “peptide-containing fraction” showed two unknown
substances assigned as stylissamide E (3) and stylissamide F (4).
The final purification of these compounds was achieved by
preparative reversed-phase HPLC.
provides more lipophilicity and therefore membrane permeability
because of a reduced zwitterionic character. Furthermore, proline
units lead to a less flexible backbone conformation and result in a
2
higher affinity and selectivity for protein binding. Examples of
proline-rich cyclopeptides are the axinastatins (7 or 8 amino acids
3
4
with 2 or 3 proline residues), axinellins (7-8 aa, 2-3 Pro),
The molecular weight of stylissamide E (3) (m/z 783.4417 [M
5
hymenamides (7-8 aa, 1-3 Pro), phakellistatins (7-10 aa, 1-4
+
+
H] ) was obtained from ESI mass spectrometry (HR-ESIMS)
6
7
Pro), and stylopeptides (7-10 aa, 2-4 Pro). Several cyclic
heptapeptides have been recently described from the marine sponge
Stylissa caribica. Stylisins 1 and 2 have been isolated from a
1
and indicated the molecular formula C39
59 8 9
H N O . The H NMR
spectrum of 3 showed seven amide protons at 8.52, 8.31, 7.93, 7.51,
1
3
7
.42, and 6.85 (2 H) ppm, while the C NMR spectrum contained
8
9
Jamaican specimen, while its Bahamanian counterpart contained
1
0
eight carbonyl signals at 174.4, 173.9, 171.4 (2 C), 170.3, 170.1 (2
C), and 168.2 ppm. The investigation of the 2D NMR spectra
identified seven amino acids. Two independent spin systems of the
the stylissamides A (1), B (2), C, and D. In addition to the four
known stylissamides, the two new proline-rich cyclic heptapeptides
stylissamides E (3) and F (4) were detected in a further specimen
of S. caribica. Herein we report the structure elucidation of 3
and 4 with a combined approach of mass spectrometric and NMR
spectroscopic techniques.
1
13
1
1
type X-CH-CH
2
-CH
2
-CH -X′ were defined using H, C-
2
1
1
1 13
HSQC-TOCSY, H, H-COSY, and H, C-HMBC and indicating
the presence of two proline units. Two spin systems of the type
X-CH-CH(CH
while the further spin systems X-CH-CH
CH -X′, and X-CH-CH -X′ were attributed to alanine, glutamine,
and tyrosine. The seven amino acids alanine, glutamine, isoleucines
3
)-CH
2
-CH
3
suggested the amino acid isoleucine,
3
, X-CH-CH
2
-
2
2
(
2×), prolines (2×), and tyrosine complied with the number of
carbonyl and amide signals of the 1D NMR data and defined the
molecule as a heptapeptide. The amino acids implied 14 degrees
of unsaturation, while the number of unsaturations indicated by
the molecular formula is 15. This difference suggested a cyclic
structure for the molecule.
The sequence of the seven amino acid residues in 3 was
1
13
elucidated by a detailed analysis of the H, C-HMBC data, which
showed correlations between the HR and the amide proton or in
the case of proline the Hδ protons and the carbonyl carbon of the
previous amino acid in the backbone of the peptide. The similar
chemical shifts of the carbonyl carbons of tyrosine and one proline
(
170.1 ppm) as well as of glutamine (171.4 ppm) and one isoleucine
1
13
were differentiated by a semiselective H, C-HMBC experiment.
The resulting correlations showed the following separated shifts in
the semiselective H, C-HMBC: 170.11 ppm for Tyr , 170.14 ppm
for Pro , 171.38 ppm for Ile , and 171.42 ppm for Gln . The
correlations Tyr NH/Pro CO and Tyr HR/Pro CO assigned the
1
13
2
3
5
6
2
1
2
1
1
2
4
3
4
3
During our search for bioactive metabolites from Caribbean
sponges, we have investigated the crude extract from specimens
of S. caribica collected in July 2000 in the Bahamas. The freeze-
dried sponge tissue was extracted with a 1:1 mixture of MeOH/
fragment Pro -Tyr , and Ala NH/Pro CO and Ala HR/Pro CO
3 4 5 4
provide the segment Pro -Ala . The correlations Ile HR/Ala CO,
6
5
7
6
Gln NH/Ile CO, and Ile HR/Gln CO elongated the second sequence
3
4
5
6
7
to Pro -Ala -Ile -Gln -Ile . The analysis of the NOESY spectrum
2
CH
2
Cl
2
, and the resulting extract was further purified by liquid/
O. The
connected both partial structures with the cross peak Tyr NH/
3
1
liquid partitioning between n-hexane, n-BuOH, and H
2
Pro HR. Further NOE cross peaks supported the sequence Pro -
2 3 4 5 6 7
Tyr -Pro -Ala -Ile -Gln -Ile for 3 (see Figure 1). The final proof
of the cyclic structure of stylissamide E (3) was achieved by analysis
of the MS/MS spectra.
*
Corresponding author. Tel: +49 471 4831 1497. Fax: +49 471 4831
1
425. E-mail: mkoeck@awi.de.
1
0.1021/np900664f  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/29/2010

Notes
Journal of Natural Products, 2010, Vol. 73, No. 4 739
a
Table 1. Chemical Shifts of Stylissamide E (3) (600 MHz)
entry
residue
Pro1
position
δ
C
/δ
N
H
δ , mult. (J/Hz)
1
2
3
4
5
6
7
8
9
N
CO
R
127
170.3
60.2
30.9
21.0
46.0
121
170.11
56.8
36.2
4.38, m
c
c
c
ꢀ,ꢀ′
γ,γ′
δ,δ′
NH
CO
R
1.99 ; 1.83
c
1.53 ; 0.72
3.19, m; 2.91, m
7.93, d (7.3)
Tyr2
b
4.08, m
3.06, dd (3.9, 13.5);
.81, t (13.2)
1
0
ꢀ,ꢀ′
2
1
1
1
1
1
1
1
1
1
2
2
2
2
1
2
3
4
5
6
7
8
9
0
1
2
3
1
127.4
115.1
129.5
156.1
2,6
3,5
4
OH
N
CO
R
6.66, d (7.5)
6.98, d (5.5)
9.27, s
Pro3
141
b
170.14
59.1
28.0
24.8
46.8
114
173.9
47.3
19.5
120
171.38
61.4
34.9
15.3
25.7
11.1
112
171.42
54.4
26.1
31.7
174.4
109
c
4.30
c
c
c
c
ꢀ,ꢀ′
γ,γ′
δ,δ′
NH
CO
R
2.19 ; 1.63
Figure 1. Backbone NOE cross peaks for stylissamide E (3).
c
1.92 ; 1.85
c
3.44 ; 3.39
The configuration of the peptide bond preceding both proline
units was established by the difference in the chemical shifts of
Ala4
7.51, d (6.8)
1
2
the ꢀ and γ carbons (∆δꢀ,γ). The differences ∆δꢀ,γ (9.9 and 3.2
ppm) indicated one cis and one trans configuration of the proline
24
2
26
4.40, m
1.36, d (7.1)
8.52, br
5
ꢀ
Ile5
NH
CO
R
1
residues in 3 (see Table 1). The similar chemical shifts of Pro HR
b
2
2
7
8
4
3
7
and Ala HR and likewise Pro HR and Ile HR caused an overlapping
3.61, dd (2.5, 7.1)
c
of the signals of the NOE spectrum, and observed correlations could
not confirm the configuration of the prolines.
The proposed sequence of the seven amino acids was verified
29
ꢀ
1.70
0.88
1
3
c
3
3
3
3
0
1
2
3
ꢀMe
γ,γ′
δ
c
c
1.56 , 1.19
c
0.86
14
by tandem mass spectrometry. The first step in the fragmentation
of a cyclic peptide is the ring-opening, which results in a mixture
of protonated linear molecules with the same molecular weight but
different sequences of amino acids. The investigation of MS/MS
experiments of cyclic peptides showed that the amide bond between
proline and the preceding amino acid is a preferred position for
Gln6
NH
CO
R
8.31, d (6.2)
b
34
35
4.02, m
c
c
3
3
3
6
7
8
ꢀ,ꢀ′
γ
1.95 ; 1.91
c
2.19
δ
c
39
40
Nꢀ
NH
CO
R
7.42, s; 6.85
1
5
Ile7
c
ring-opening. The second step is the fragmentation of the linear
structure of the peptide, generally by successive loss of the C- or
N-terminal amino acid in the MS/MS experiments. The isolation
110
6.85
4
4
4
4
1
2
3
4
168.2
54.3
36.6
15.8
24.3
11.9
4.32, m
c
ꢀ
1.83
n
and fragmentation of the resulting MS/MS ions in MS experiments
ꢀMe
γ,γ′
δ
0.95, d (6.5)
c
c
give additional information about the sequence of the amino acids.
The MS/MS fragmentation of 3 showed the loss of the amino
acids alanine, isoleucine, glutamine, and proline directly from the
parent ion and proved the cyclic structure of the heptapeptide with
a possible ring-opening at several positions. The differentiation of
leucine and isoleucine resulted from the NMR spectra, in which
only the spin systems for isoleucine were observed. The main
fragmentation pathway of m/z 670.3865, 542.3615, 429.2596, and
45
1.50 ; 1.09
0.85
c
4
6
a
1H and 13C chemical shifts are referenced to the DMSO-d signal
6
1
5
(2.50 and 39.5 ppm, respectively).
calibrated with an external standard. Therefore, the δ value has an
accuracy of about 1 ppm in reference to NH
chemical shifts are given without decimals.
carbonyl carbons are given with two decimals if one decimal did not
N chemical shifts were not
15
3
(0 ppm), and the
N
b
13
C
NMR shifts of
c
allow for differentiation of two different carbonyl carbons. Overlapped
3
58.2480 showed the losses of isoleucine, glutamine, isoleucine,
signals.
and alanine. These results confirmed the proposed amino acid
sequence with a ring-opening between Ile and Pro and an
7
1
The H and 13C NMR spectra of 4 showed five amide signals at
8.25, 7.59, 7.45, 7.38, and 7.35 ppm and eight carbonyl signals at
172.7, 171.9, 171.1, 171.0, 170.8 (2 C), 170.4, and 168.5 ppm (see
Table 3). 2D NMR techniques assigned seven amino acids including
two different types of spin systems. The repeated existence of the
1
7
6
5
4
elimination of the single amino acids Ile , Gln , Ile , and Ala from
the C-terminal end of the peptide (see Figure 2 and Table 2). A
3
3
weak signal for the loss of Pro extended this sequence to Pro -
4
5
6
7
Ala -Ile -Gln -Ile . Besides the main fragments, there were ions
with a reduced molecular weight of m/z 17, 28, and 45, which
correspond to the elimination of ammonia, carbon monoxide, and
spin system X-CH-CH
and one arginine residue within the peptide, while X-CH-CH
2
-CH
2
-CH
2
-X′ indicated three proline
2
-X′
both together (NH
3
+ CO) from the main fragments (for example:
were attributed to two phenylalanine units and one aspartic acid.
A difference in the degree of unsaturation s 19 in the amino acids
and 20 in the calculated molecular formula s suggested a cyclic
structure for the heptapeptide. The sequencing of the amino acids
was complicated due to the overlap of two proline HR signals and
similar chemical shifts for some carbonyl carbons. The amide proton
and HR region of the semiselective H, C-HMBC experiments
showed seven correlations between the HR or the amide proton
and the carbonyl carbon of the previous amino acid. The signals
m/z 765.4238, 755.4285, and 738.4422). The fragment at m/z
4
5
82.3304 indicated the loss of the amino acids Ile5 and Ala
combined with an elimination of ammonia. The fragment m/z
3
4
29.2596 lost proline and alanine under MS conditions. The
4
1
C-terminal elimination of Ala and N-terminal elimination of Pro
determined the sequence Pro -Tyr -Pro -Ala . The sequence of 3
1
2
3
4
1
13
was finally established by a combined approach of NMR and mass
1
2
3
4
5
6
spectrometric data as cyclo-(cis-Pro -Tyr -trans-Pro -Ala -Ile -Gln -
Ile ).
7
3
2
4
3
5
4
6
Phe HR/Pro CO, Asp HR/Phe CO, Pro HR/Asp CO, Arg HR/
5 7 6 2 3
The molecular weight of stylissamide F (4) (m/z 857.4266 [M
Pro CO, and Phe HR/Arg CO determined the segment Pro -Phe -
4 5 6 7 4 3
+
+
H] , HR-ESIMS) indicated the molecular formula C43
57 10 9
H N O .
Asp -Pro -Arg -Phe , while the correlations Asp NH/Phe CO and

7
40 Journal of Natural Products, 2010, Vol. 73, No. 4
Notes
Figure 2. HR-MS/MS spectrum of stylissamide E (3). The precursor ion is marked with a solid square. Only the main fragmentation
pathway is indicated.
n
a
Table 2. Fragments Observed in HR-MS/MS and MS Experi-
Table 3. Chemical Shifts of Stylissamide F (4) (600 MHz)
ments on Stylissamide E (3)
entry
residue
Pro1
position
δ
C
/δ
N
H
δ , mult. (J/Hz)
3
m/z
formula
sequence
MS loss
1
2
3
4
5
6
7
8
9
N
CO
R
ꢀ,ꢀ′
γ,γ′
δ,δ′
N
141
170.4
59.2
27.7
24.5
46.3
127
170.82
59.8
30.6
20.7
45.8
122
171.1
57.2
2
61.1
58.2
29.3
71.3
42.4
C
C
C
C
C
14
19
22
25
28
H
H
H
H
H
17
24
29
35
40
N
N
N
N
N
2
3
4
4
5
O
O
O
O
O
3
4
5
5
6
Pro-Tyr/Tyr-Pro
Pro-Tyr-Pro
Pro-Tyr-Pro-Ala
Ile-Pro-Tyr-Pro
Ile-Pro-Tyr-Pro-Ala/
Pro-Tyr-Pro-Ala-Ile
Pro-Tyr-Pro-Ala-Ile-Gln
c
4.30
3
4
4
5
Pro
2.16, m; 1.66, m
Ala, Pro
c
1.91, m; 1.84
3.50, m; 3.36
c
Ile, Ala
Pro2
b
CO
R
6
70.4
C
H
33 48
N
7
O
8
Pro, Gln
c
4.31
c
1
0
ꢀ,ꢀ′
γ,γ′
δ,δ′
NH
CO
R
2.02, m; 1.82
7
6
11
1.51, m; 0.62, m
Phe NH/Arg CO confirmed this sequence. The similar chemical
c
1
1
2
3
3.17 ; 2.81, t (10.0)
1
2
shifts of Pro HR and Pro HR caused an overlap of the potential
Phe3
8.25, br
2
1
1
2
signals Pro HR/Pro CO and Pro HR/CO, but the correlation Pro Hδ/
Pro CO elongated the proposed fragment to Pro -Pro -Phe -Asp -
Pro -Arg -Phe . A weak correlation existed between Pro Hδ and
Phe CO, which indicated the ring-closure of 4.
14
15
16
1
1
2
3
4
4.12, m
c
5
6
7
1
ꢀ,ꢀ′
1
2,6
3,5
4
36.5
3.15 ; 3.09, d (12.8)
1
1
7
8
137.2
128.5
128.1
126.5
109
170.83
48.5
7
c
7.20
7.25
7.21
1
2
5
c
The ∆δꢀ,γ values (3.2, 9.9, and 4.0 ppm for Pro , Pro , and Pro )
indicated a cis configuration for the peptide bond preceding Pro
19
20
21
2
c
Asp4
Pro5
Arg6
NH
CO
R
7.59, d (4.5)
1
5
and a trans configuration for Pro and Pro (see Table 3). Similar
b
22
23
24
1
2
chemical shifts of Pro HR and Pro HR caused an overlapping of
the signals with the diagonal of the NOESY spectrum.
4.72, m
c
ꢀ,ꢀ′
γ
34.4
172.7
3.21 ; 2.89, d (15.1)
Tandem mass spectrometry data verified the NMR results. The
main fragmentation pathway of 4 under MS/MS conditions implied
the loss of C-terminal and N-terminal amino acids. The cyclic
structure of stylissamide F (4) was confirmed by the loss of aspartic
acid, phenylalanine, and proline after ring-opening at different amide
bonds directly from the parent ion of 4. The presence of three
proline and two phenylalanine residues in the heptapeptide com-
plicated the sequence elucidation of the single fragments with MS/
25
26
27
28
29
OH
N
132
CO
R
171.9
63.3
28.9
24.9
46.9
110
171.0
53.3
27.3
25.0
40.1
85
4.04, m
30
ꢀ,ꢀ′
γ,γ′
δ,δ′
NH
CO
R
2.23, m; 1.71, m
c
31
32
33
34
2,01, m; 1,83
3.77, m; 3.70, m
7.38, br
n
MS and MS experiments. For example, the fragment m/z 401.2412
35
36
37
3.98, m
5
6
7
6
7
1
was assigned as Pro -Arg -Phe and as Arg -Phe -Pro due to the
ꢀ,ꢀ′
γ,γ′
δ,δ′
Nꢀ
ꢁ
1.65, m; 1.56, m
1.39, m; 1.31, m
3.05, m
5
6
7
1
loss of N- and C-terminal prolines from Pro -Arg -Phe -Pro .
3
3
8
9
2
3
4
3
4
Similarly, m/z 360.2310 indicated Pro -Phe -Asp and Phe -Asp -
c
7.45
5
2
3
4
5
Pro , both secondary fragments of Pro -Phe -Asp -Pro . In addition
40
41
42
to proline as a common position for ring-opening 4 contained the
Nη
NH
CO
R
Phe7
114
7.35, br
4.69, m
4
5
sequence Asp -Pro . Protonation of the proline amide with the acidic
proton of aspartic acid caused the preference for a cleavage of this
amide bond. A cyclization of the aspartic acid side chain with the
43
44
45
168.5
51.1
36.6
c
ꢀ,ꢀ′
3.22 ; 2.76 dd
1
6
new C-terminal carbonyl group possibly reduced the C-terminal
(6.3, 14.0)
4
46
47
1
137.9
129.3
127.6
125.6
fragmentation of the peptide. After ring-opening at position Asp
5
5
6
2,6
3,5
4
7.30, d (5.5)
and Pro , the precursor ion successively lost N-terminal Pro , Arg ,
c
4
4
8
9
7.23
7.18
7
1
4
Phe , and Pro and C-terminal Asp and gave the fragments m/z
c
760.5267, 604.4318, 457.2371, 360.2310, and 245.1250 (see Figure
a
1H and 13C chemical shifts are referenced to the DMSO-d
6
signal
N chemical shifts were not
3
). The signals at m/z 254.1376 and 195.0779 indicated the
1
5
5
6
1
2
(2.50 and 39.5 ppm, respectively).
calibrated with an external standard. Therefore, the δ value has an
accuracy of about 1 ppm in reference to NH
chemical shifts are given without decimals.
carbonyl carbons are given with two decimals if one decimal did not
fragments Pro -Arg and Pro -Pro . The elimination of ammonia,
carbon monoxide, and both together from the main fragments was
also observed (for example: m/z 840.4581, 829.4737, and 812.4870).
15
3
(0 ppm), and the
N
b
13
C
NMR shifts of
1
2
3
4
The fragment m/z 457.2371, assigned as Pro -Pro -Phe -Asp and
2
3
4
5
3
c
Pro -Phe -Asp -Pro , lost proline and aspartic acid under MS
conditions. The sequence of 4 was finally established by a combined
allow for differentiation of two different carbonyl carbons. Overlapped
signals.

Notes
Journal of Natural Products, 2010, Vol. 73, No. 4 741
Figure 3. HR-MS/MS spectrum of stylissamide F (4). The precursor ion is m/z 857.4283. Only one possible fragmentation pathway is
indicated.
approach of NMR and mass spectrometric data as cyclo-(trans-
Pro -cis-Pro -Phe -Asp -trans-Pro -Arg -Phe ).
the collections of the Zoological Museum of Amsterdam under reg.
no. ZMA POR. 15607.
1
2
3
4
5
6
7
Extraction and Isolation. The freeze-dried sponge tissue of S.
caribica (50.09 g) was crushed with a mill and extracted exhaustively
at room temperature with a 1:1 mixture of MeOH/CH Cl . The crude
The absolute configuration of the amino acid residues of
1
7
stylissamides E and F (3 and 4) was determined by Marfey′s and
1
8
2
2
the OPA method after hydrolysis of 3 and 4. All amino acids
extract (23.29 g) was partitioned between n-hexane (4 × 400 mL) and
had the L-configuration.
MeOH (300 mL). The MeOH extract was then partitioned between
n-BuOH (5 × 300 mL) and H
2
O (300 mL). The resulting n-BuOH
Conclusion
phase (11.56 g) from the solvent partitioning scheme was purified by
gel chromatography on Sephadex LH-20 (Pharmacia) using MeOH as
the mobile phase. The final purification of the isolated compounds was
achieved by preparative RP18 HPLC on a Prontosil Eurobond C18
column (20 × 250 mm, 5 µm) applying a MeCN/TFA (0.1%) gradient
to afford 3 (2.0 mg, 0.004% of dry weight) and 4 (0.7 mg, 0.001% of
dry weight).
The family of cyclic heptapeptides from the sponge S. caribica
is extended by the stylissamides E (3) and F (4). Stylissamide E
(
3) includes the segment Ile-Pro-Tyr-Pro, which is known from
stylisin 2 as well as stylissamides C and D. The amino acids alanine
and glutamine in 3 rarely appear in this family of peptides.
Stylissamide E (3) is the first member of this family with only two
proline residues. Compared with the other stylissamides, stylissa-
mide F (4) is a rather polar peptide due to the coexistence of the
basic amino acid arginine with aspartic acid. As in stylissamides
A (1), C, and D, stylissamide F (4) has three proline residues,
including two successive prolines as in stylissamide A (1). The
structure elucidation of the new cyclic peptides was established by
a combined approach of mass spectrometry and NMR spectroscopy.
Stylissamide E (3): light yellow powder; UV (DAD) λmax ) 221,
1
2
80 nm; CD (1.1 mg/10 mL, MeOH) λmax (∆ε) 222 nm (-1.035); H
13
and C NMR data in Table 1; HPLC/HR(+)ESI-MS t
R
) 16.1 min,
+
m/z ) 783.4417 [M + H] (calcd for C39
.1 ppm.
Stylissamide F (4): light yellow powder; UV (DAD) λmax ) 278
H N O , 783.4400), ∆m )
59 8 9
2
1
nm; CD (0.3 mg/5 mL, MeOH) λmax (∆ε) 218 nm (-0.670); H and
1
3
C NMR data in Table 2; HPLC/HR(+)ESI-MS t
R
) 17.2 min, m/z
+
)
857.4266 [M + H] (calcd for C43
H N O , 857.4304), ∆m ) 4.4
57 10 9
ppm.
Experimental Section
General Experimental Procedures. H, 13C, and 15N NMR spectra
were conducted on a Bruker Advance 600 MHz NMR spectrometer.
Determination of the Absolute Configuration of Proline by a
1
17
Modified Marfey’s Method: Stylissamides E and F (3 and 4, 600
µg each) were placed in 1 mL conical vials containing HCl (16%, 0.5
6
All experiments were measured at 303 K with DMSO-d as solvent.
mL), and the sealed vials were heated at 100 °C for 12 h. After
1
1
1
13
1
13
1
13
The DQF- H, H-COSY, H, C-HSQC, H, C-HSQC-TOCSY, H, C-
2 2
evaporation of the solvent under N , H O (100 µL) was added. A 40
1
13
HMBC, semiselective H, C-HMBC (pulse: Gaus1.1000, pulse length:
µL aliquot of this solution was used for the OPA method. To the
remaining hydrolysis solution (60 µL) were added NaHCO (0.1 M,
00 µL) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (L-FDAA.
1
15
1
15
1
.4 ms, spectral range: 165-178 ppm), H, N-HSQC, H, N-HMBC,
1 1
3
and H, H-NOESY experiments were carried out using standard
parameters. The mixing time for NOESY spectra was 200 ms, and the
delay for the H, C-HMBC experiment was 80 ms. HPLC-MS analysis
were performed with an Agilent 1100 HPLC system and Bruker
Daltonics microTOFLC. Analytical chromatography: Waters XTerra RP18
1
1
13
0.1%, 50 µL) in acetone, and the sealed vials were heated at 80 °C for
5
5
min. To the reaction mixture were added HCl (0.2 M, 50 µL) and
0% aqueous MeCN (containing 0.1% HCOOH, 90 µL). The mixture
was subjected to HPLC analysis [Waters XTerra RP18 column (3.0 ×
50 mm, 3.5 µm); MeCN/H O/HCOOH gradient: 0 min: 30% MeCN/
70% HCOOH (0.01%); 30 min: 60% MeCN/40% HCOOH (0.01%)
column (3.0 × 150 mm, 3.5 µm) with a MeCN/H
2
O/HCOOH gradient
1
2
[
0 min: 10% MeCN/90% HCOOH (0.01%); 30 min: 60% MeCN/40%
-
1
HCOOH (0.01%) with a flow rate of 0.4 mL min ]. Preparative
chromatography: Prontosil Eurobond C18 column (20 × 250 mm, 5
µm) with a MeCN/TFA (0.1%) gradient. UV spectra were recorded
during HPLC analysis with a DAD (Agilent). ESIMS/MS spectra were
recorded with an Exquire 3000plus ion trap (Bruker Daltonics).
Animal Material. The marine sponge Stylissa caribica was collected
by scuba diving on July 29 and 30, 2000, at Little San Salvador in the
Bahamas (75 to 91 ft depth). The samples were immediately frozen
after collection and kept at -20 °C until extraction. The sponge material
-
1
with a flow rate of 0.4 mL min ]. UV detection was performed at a
wavelength of 340 nm.
Determination of the Absolute Configuration of Amino Acids
1
8
by the OPA Method: A 40 µL aliquot of the hydrolysis solutions
was used for the OPA method. In an HPLC vial, 80 µL of o-
phthaldialdehyde (OPA) solution and 80 µL of N-isobutyrylcysteine
(0.1%) were added to this solution, and after a reaction time of 2 min
20 µL of the reaction mixture was subjected to HPLC analysis
[Phenomenex Hyperclone BDS C18 column (4.0 × 250 mm, 5 µm);
1
9
was compared with previously investigated material of S. caribica
and was found to match closely (Dr. M. Assmann, personal com-
munication). A detailed taxonomic description of the sponge is given
in ref 20, and a voucher fragment of S. caribica has been deposited in
2
MeOH/NaOAc gradient: solution A: 125 mM NaOAc in H O and 20
mL of MeOH, adjusted to pH 6.8 using diluted AcOH solution B:
MeOH].

7
42 Journal of Natural Products, 2010, Vol. 73, No. 4
Notes
Acknowledgment. Financial support from the Deutsche Forschungs-
gemeinschaft (DFG) under grant Ko 1314/5-1 (DFG research unit FOR
34) is gratefully acknowledged. Sponge collection was carried out by
(5) (a) Kobayashi, J.; Tsuda, M.; Nakamura, T.; Mikami, Y.; Shigemori, H.
Tetrahedron 1993, 49, 2391–2402. (b) Tsuda, M.; Shigemori, H.; Mikami,
Y.; Kobayashi, J. Tetrahedron 1993, 49, 6785–6796. (c) Tsuda, M.;
Sasaki, T.; Kobayashi, J. Tetrahedron 1994, 50, 4667–4680. (d) Koba-
yashi, J.; Nakamura, T.; Tsuda, M. Tetrahedron 1996, 52, 6355–6360.
9
Dr. M. Assmann during a scientific expedition to the Bahamas in 2000.
During this time the project was sponsored by the DFG (Ko 1314/3-1
to 3-4). We would like to acknowledge the support of Prof. Dr. J. R.
Pawlik (University of North Carolina, Wilmington), who gave members
of the K o¨ ck research group the opportunity to participate in scientific
sojourns to the Bahamas (1998 to 2008). We further thank E. Lichte
for performing preparative HPLC analysis, J. Scholz (AG Prof. Dr.
M. Reggelin, TU Darmstadt) for measuring the CD spectra and K.-U.
Ludwichowski (AG Prof. Dr. G. Kattner) for carrying out the amino
acid analysis (for non-proline residues).
(
(
6) Pettit, G.; Tan, R. J. Nat. Prod. 2005, 68, 60–63.
7) (a) 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. (b) Brennan, M. R.; Costello, C. E.;
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4
53–456.
8) Mohammed, R.; Peng, J.; Kelly, M.; Hamann, M. J. Nat. Prod. 2006,
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(
(
6
9) Stylissa caribica collected on July 28, 2000, at Little San Salvador in
the Bahamas (74 ft depth).
(
(
10) Schmidt, G.; Grube, A.; Köck, M. Eur. J. Org. Chem. 2007, 4103–4110.
11) Stylissa caribica collected on July 29 and 30, 2000, at Little San
Supporting Information Available: HPLC-MS spectrum of Stylissa
caribica; NMR and CD spectra of compounds 3 and 4. This material
is available free of charge via Internet at http://pubs.acs.org.
Salvador in the Bahamas (75 to 91 ft depth).
(12) Siemion, I. Z.; Wieland, T.; Pook, K.-H. Angew. Chem., Int. Ed. Engl.
975, 14, 702–704.
(13) W u¨ thrich, K.; Billeter, M.; Braun, W. J. Mol. Biol. 1984, 180, 715–740.
1
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NP900664F