Callyaerins A-F and H, new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa

Article abstract of DOI:10.1016/j.bmc.2010.06.012

Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new cytotoxic cyclic peptides callyaerins A-F (1-6) and H (8). Their structures were determined using extensive 1D ( ¹H, ¹³C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5-9 amino acids and side chains of 2-5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the template for ring closure and afforded the linkage to the peptidic side chain which was always initiated with a proline moiety. All peptides contained three or more proline residues and the remaining residues were predominantly hydrophobic residues with all amino acids present in the l form. Callyaerins A-F (1-6) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited strong activity against the L5178Y cell line with ED₅₀ values of 0.39 and 0.48 μM, respectively. On the other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans.

Full text of DOI:10.1016/j.bmc.2010.06.012

Bioorganic & Medicinal Chemistry 18 (2010) 4947–4956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Callyaerins A–F and H, new cytotoxic cyclic peptides from the Indonesian
marine sponge Callyspongia aerizusa
Sabrin R. M. Ibrahim a, , Cho Cho Min a, Franka Teuscher a,à, Rainer Ebel b, Christel Kakoschke c, Wenhan Lin d,
e,
a,
*
Victor Wray c, , RuAngelie Edrada-Ebel , Peter Proksch
*
*
a Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität, Geb. 26.23, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
b Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Building, Meston Walk, Old Aberdeen, AB24 3UE, Scotland, UK
c Department of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany
d State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Xueyuan Road, 38, Beijing 100083, China
e Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new
cytotoxic cyclic peptides callyaerins A–F (1–6) and H (8). Their structures were determined using exten-
sive 1D (1H, 13C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI
and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5–9 amino acids
and side chains of 2–5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the
template for ring closure and afforded the linkage to the peptidic side chain which was always initiated
with a proline moiety. All peptides contained three or more proline residues and the remaining residues
Received 22 March 2010
Revised 26 May 2010
Accepted 4 June 2010
Available online 11 June 2010
Dedicated to the memory of Cho Cho Min
were predominantly hydrophobic residues with all amino acids present in the
(1–6) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays
employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited
L form. Callyaerins A–F
Keywords:
Callyspongia aerizusa
Callyaerins
strong activity against the L5178Y cell line with ED50 values of 0.39 and 0.48
other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans.
Ó 2010 Elsevier Ltd. All rights reserved.
lM, respectively. On the
Proline-rich cyclic peptides
Cytotoxicity
Antibacterial
Antifungal
1. Introduction
around 15 species only have been investigated chemically. Sponges
belonging to the genus have been known to yield biologically
active polyacetylenic10 and nitrotetradecenyl pyridine11 com-
pounds with different chain lengths and unsaturation positions.
Nematocidal depsipeptides, phoriospongin A and B were isolated
from the Australian marine sponge Callyspongia bilamellata.9 To
date only our previous work describing a preliminary investigation
of the metabolic diversity of the Indonesian sponge Callyspongia
aerizusa has provided an insight into the chemical potential of this
sponge species. We were able to isolate a new cytotoxic cyclic pep-
tide callyaerin G (7)12 and found indications of a considerable
number of other cyclic peptides present in the extract. These pep-
tides were related analogues of callynormine A, a cyclic peptide
isolated from the southern Kenyan sponge Callyspongia abnormis.13
As a continuation of this work we have now completed a detailed
investigation of the bioactive ethyl acetate fraction of this sponge.
In addition to the callyaerin G (7) previously described by us,12
here we describe the isolation, structure elucidation, and biological
activities of a further seven callyaerin congeners of variable ring
size and side-chain length. Most of the isolated compounds dis-
played strong activity against the tumour cell line L5178Y, and
varying degrees of antibacterial and antifungal properties.
Marine sponges are a rich source of bioactive cyclic peptides
and depsipeptides with unique structures involving a wide variety
of unusual amino acids and other building blocks.1–4 Structure elu-
cidation of such compounds can be quite challenging insofar as
they defy traditional amino acid sequencing strategies. Many cyclic
peptides isolated from marine sponges exhibit interesting biologi-
cal properties including a reverse of multi-drug resistance in
tumour cells,5 cytotoxicity,6 HIV inhibition,7,8 and nematocidal
activity.9 The genus Callyspongia includes 182 species, of which
* Corresponding authors. Tel.: +49 211 81 14163; fax: +49 211 81 11923 (P.P.);
tel.: +49 531 6181 7200; fax: +49 531 6181 7099 (V.W.); tel.: +44 (0)141 548 3924;
fax: +44 (0)141 552 2562 (R.E.-E.).
E-mail addresses: victor.wray@helmholtz-hzi.de (V. Wray), ruangelie.edrada-
ebel@strath.ac.uk (RuAngelie Edrada-Ebel), proksch@uni-duesseldorf.de (P.
Proksch).
Permanent address: Pharmacognosy Department, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt.
à
Present address: Malaria Drug Resistance and Chemotherapy Laboratory, The
Queensland Institute of Medical Research, 300 Herston Rd., Herston, Brisbane,
Queensland 4006, Australia.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.012

4948
S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
eronuclear NMR techniques, in combination with ESI-MS and ami-
2. Results and discussion
no acid analysis, to provide unambiguous structures of the various
compounds. Initially we chose callyaerin E (5, Fig. 2) as a reference
compound, since it was available in sufficient quantity to allow the
acquisition of a complete set of 1D and 2D NMR spectra. These
provided exemplary data from which we were able to develop a
strategy to elucidate the structures of the remaining analogues
some of which were isolated in low yields precluding the recording
of a full set of spectra.
The EtOAc soluble extract of the sponge C. aerizusa was fraction-
ated using silica gel column chromatography and the resulting
fractions were subjected to MTT assays toward the tumour cell
lines L5178Y, HeLa, and PC12. The active fractions were subjected
to semi-preparative HPLC to afford fractions containing seven
novel cyclic peptides that were sufficiently pure to allow structural
studies. In all cases we have used a combination of homo- and het-
Table 1
Complete 1H and 13C NMR data of callyaerin E (5) (DMSO-d6, 600 MHz)
A. A.
DAA
Position dC
NH
dH (J in Hz)
ROESYa
1- , 1-NH, 7-NH, 7-
HMBCa
8.36 s
a
a, 7-
c
, C1-
a
, C1-d 7-
a
, 7-CO, DAA–CO
CO
a
167.5 s
98.8 s
b
143.3 d
7.28 d (13.6)
1-
a
, DAA-a, DAA–CO
R1 Leu NH
5.27 dd (13.3, 10.0)
DAA–NH
1-CO, DAA-
a
CO
170.8
a
b
57.9 d
41.8 t
4.36 m
1.77 m, 1.23 m
1.60 m
2-
a
, 2-b, 2-d, DAA–NH
1-CO, 8-
1-CO
a
c
26.5 d
22.5 q, 23.2 q
171.9 s
63.9 d
29.1 t
1-a
d, d0
0.96 d (6.0), 0.94 d (6.6)
1-b
R2 Pro CO
a
b
c
d
4.01 dd (9.9, 5.5)
2.16 m, 1.67 m
2.06 m, 1.88 m
3.93 br t (8.8) 3.59 m
7.73 d (6.7)
1-
1-
3-NH
a
a
, 3-a, 3-NH
1-CO, 2-CO, 2-
c
, 3-NH
24.9 t
47.8 d
R3 Phe NH
2-
4-
a
a
, 2-b, 2-
c
, 4-NH, 4-
a
2-CO, 2-
a
, 3-
a
, 3-b, 3-CO
CO
a
b
168.9 s
54.1 d
35.1 t
4.39 m
3.00 m
, 4-NH
3-a
, 3-CO, 100, 200
Aromatic 126.3, 128.5, 138.3
R4 Phe NH
CO
7.17–7.32 m
7.31 d (6.9)
3-NH
3-CO, 4-b, 4-CO
171.2 s
53.1 d
36.1 t
a
b
4.63 ddd (8.8, 6.9, 4.7)
3.00 m, 2.71 dd (13.3, 4.7) 5-
3-
a
a
, 3-b, 5-d
3-CO, 4-b, 4-CO
5-a
, 5-CO, 10, 20, 60
, 6-
c
Aromatic C1: 137.3 s, C2,6: 129.5 d, C3,5: 129.1 d, C4: 126.5 d 1 7.17–7.30 m
R5 Pro CO
171.3 s
62.8 d
28.4 t
24.5 t
47.2 d
171.5 s
64.0 d
29.6 t
25.8 t
49.5 d
a
b
c
d
4.41 m
6-
a
4-CO
2.15 m, 1.82 m
1.93 m, 1.89 m
3.49 br t (8.8), 3.42 m
4-
3-
a
a
4-CO
R6 Pro CO
a
b
c
d
4.16 dd (7.9, 9.8)
2.22 m, 1.41 m
1.74 m, 1.59 m
3.34 m, 2.48 m
7.03 d (10.1)
5-
a
, 7-
a
, 7-NH
5-CO, 6-CO
5-CO
R7 Val NH
DAA–NH
6-a
, 7-a, 7-b, 7-CO
CO
a
171.2 s
56.5 d
4.56 dd (10.1, 3.9)
2.33 m
6-
a
, 6-b, 7-
c
, DAA–NH
7-b, 7-c, 7-CO
b
28.8 d
c
18.4 q, 18.3 q
172.4 s
59.5 d
1.06 d (6.9), 0.97 d (6.8)
C1 Pro CO
a
4.36 m
2-
2-
a
a
, 2-NH
, 2-NH
2-a
b
c
d
26.6 t
23.8 t
45.8 d
2.22 m, 1.57 m
1.78 m, 1.68 m
3.57 m, 3.21 m
7.62 d (7.9)
2-
1-
a
a
, 2-NH, DAA–NH
, 1-b, 1-d, 3-
1-b, 1-CO, 1-
1-a, 1-CO, 2-
c
a
C2 Ile NH
a, 3-NH
, 2-CO
CO
a
b
171.1 s
58.3 d
35.3 d
3.99 dd (7.9, 7.3)
1.95 m
1-
a
, 1-b, 1-d, 3-
a, 3-NH
2-b, 2-CO, 2-
c
c
c
24.6 t
15.9 q, 11.4 q
1.45 m, 1.21 m
0.88 d (6.9), 0.85 t (6.9)
7.37 d (8.0)
d
C3 Ile NH
2-NH, 2-
a
, 2-d, 2-
c
, 4-NH
, 4-NH
2-
a
, 3-
a
, 3-CO
CO
a
170.8 s
57.8 d
4.08 t (7.6)
2-
a
, 2-NH, 2-b, 2-
c
3-b, 3-d, 3-c
, 3-CO
b
34.2 d
1.82 m
4-NH
c
c
25.1 t
15.0 q, 10.8 q
1.39 m, 1.27 m
0.87 d (6.9), 0.80 t (6.7),
7.88 t (6.0)
d
C4 Gly NH
Terminal-NH2, 3-NH, 3-
3- , 3-d
4-NH, 4-
a, 3-b
3-a, 3-CO, 4-CO, 4-a
CO
a
170.9 s
42.3 t
3.72 dd (16.7, 6.2)
3.57 dd (16.7, 5.9)
7.04 br s, 6.97 br s
a
4-CO
NH2
a
4-CO, 4-a
a
The numbering refers to residues in the ring for R-residues and in the chain for C-residues.

S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
4949
terminal NH2 group of the peptidic side chain, as there were no
Gln or Asn units present.
Ring
Sidechain
C1 C2 C3 C4 C5
Pro Ile Phe Gly
Pro Ile Ile
R1 R2 R3 R4 R5 R6 R7 R8
Ile Hyp Val Ile Leu Pro Pro Leu
Ile Hyp Ile Ile Leu Pro Pro Leu
His Hyp Leu Leu Pro Pro Val
Ile Ile Phe Pro Hyp Pro Leu
Leu Pro Phe Phe Pro Pro Val
Val Pro Val Phe Pro
In the 13C spectrum the presence of 12 carbonyls at dC 167.7–
172.7 ppm and 11
a-carbons of amino acids in the region dC
1
2
3
4
5
6
7
8
A
B
C
D
E
F
42.3–58.3 further confirmed a compound featuring 11 amino acids
along with a DAA unit. Thus, 25 of the 26 double bond equivalents
required by the molecular formula were accounted for by the car-
bonyl groups, proline rings, DAA, and aromatic rings of Phe, indi-
cating 5 was a cyclic peptide. The sequence of amino acids and
the location of DAA were established by a detailed examination
of the ROESY and HMBC spectra (Fig. 3). In the ROESY spectrum
Pro Leu Phe Gly
Pro Ile Asn Ala Ile
Pro Ile Ile Gly
Pro Leu Phe Ile
Pro Phe Phe Phe
Pro Ile
diagnostic NOE correlations (Table 1) were observed between H
a
and NH of adjacent residues for residues R2–R4, R6–DAA and
C1–C4, and between NH and NH of adjacent residues for residues
R7–DAA–R1, R3–R4, and C2–C4. The positions of the Pro residues
G
H
Leu Pro Pro Pro Pro Leu
Val Pro Val Phe Pro Pro Leu
R2 and R5 were apparent from the correlations of H of the preced-
ing residue with Hd’s of these Pro units. The position of both the
closure of the ring system and the attachment of the side chain fol-
a
Footnote: Hyp is γ-hydroxyproline.
lowed from the correlations of the NH of DAA (Table 1) to both H
a
R7
R8
NH
R6
R3
and NH of R1 and to H and Hd of C1, respectively. The location of
a
O
H
NH
CO
R5
R4
the side chain terminal NH2 was determined from the correlations
of NH2 to NH and H of C4. Finally the sets of NOE correlations be-
a
C1
C4 C3 C2
C5
H2N
tween signals of R1 and C1 with those of DAA defined the (Z)-con-
figuration of the DAA unit (Fig. 1) which was the same as that
found in the X-ray structure of callynormine A.13
The sequence established from the through-space homonuclear
NMR data was confirmed from the through-bond heteronuclear
correlations observed in the HMBC spectra (Table 1). In particular
the two substructures Leu-Pro-Phe-Phe-Pro-Pro-Val (R1–R7) and
Pro-Ile-Ile-Gly (C1–C4) were corroborated by HMBC correlations
R1 R2
NH
CO
Figure 1. Structure of all the compounds isolated from Callyspongia aerizusa. The
variable size of the ring and side-chain residues of the cyclic peptides necessitated
the use of the nomenclature shown where R refers to ring residues and C to side-
chain residues. The structure elucidation of callyaerin G has been reported by us
elsewhere.12
of NH and H of each amino acid to the amide carbonyl carbons
a
2
3
through JCH and JCH. In the case of the ring Pro-units the respec-
Callyaerin E (5) was obtained as a white amorphous powder.
Positive mode ESIMS showed pseudo-molecular ion peaks at m/z
1262 [M+H]+ and 1284 [M+Na]+, which were consistent with the
molecular formula C66H95N13O12 as confirmed by HRESI-TOFMS.
Extensive analysis of 2D NMR data (TOCSY and COSY) disclosed
the presence of eleven spin systems which could be assigned to
eleven amino acid residues. Seven characteristic spin systems in
the TOCSY spectrum were detected to low field (7.9–5.2 ppm)
and originated from the amidic protons of one Gly, one Leu, two
Ile, two Phe and one Val. [See Figs. S1–S3 of Supplementary data.]
Close inspection of the high field region indicated the presence of
four Pro units. In addition, the signals at dH 8.36 (s, NH) and 7.28
(d, J = 13.6 Hz, b-H) in association with their HMBC correlations
to dC 167.7 (s), 98.8 (s), and 143.3 (d) indicated the presence of
an unusual (Z)-2,3-diaminoacrylic acid (DAA) moiety.12,13 Two
singlet signals at dH 7.04 (br s) and 6.97 (br s) were assigned to a
tive H always correlated with the carbonyl carbon of the preced-
ing unit and in most cases a correlation with the same carbonyl
carbon was observed for Hd (Table 1). The position of the DAA unit
a
was confirmed from HMBC correlations of b-H (DAA) to C (R1)
a
and in reverse NH (R1) to C (DAA). A complete assignment of
a
the 1H and 13C NMR data is given in Table 1. Initially it was partic-
ularly difficult to distinguish Leu and Ile as these occurred in con-
siderable numbers in all compounds. However a careful inspection
of the multiplicity of H in the well resolved 1D 1H spectra and the
a
characteristic 13C chemical shifts of the
afforded unambiguous assignments.
c- and d-methyl groups
The base peak at m/z 865 [Mꢀ(Pro+2Ile+Gly+NH2)]+ for com-
pound 5, arises from cleavage of the side chain. As is usual with
this type of molecule the fragmentation patterns in the ESI-MS
and HRESI-TOFMS spectra are of limited use in providing sequence
information as internal fragmentation of the ring system is difficult
Ile-11
Val-7
H
Pro-6
N
O
O
H
N
O
O
H
H
N
H
N
N
O
O
N
H
H2N
N
O
O
Gly-12
Z-DAA
Pro-5
Phe-4
N
NH
O
Ile-10
Pro-9
O
O
O
NH
N
N
H
Leu-1
Pro-2
Phe-3
Figure 2. Structure of callyaerin E (5).

4950
S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
Table 2
Table 2 (continued)
1H and 13C NMR data of Callyaerin A (1) (DMSO-d6, 600 MHz).
A. A.
Position
dC
dH (J in Hz)
A. A.
DAA
Position
dC
dH (J in Hz)
C4 Gly
NH
CO
a
8.15 dd (6.8, 5.5)
NH
CO
a
8.29 s
170.8 s
42.0 t
167.7 s
98.2 s
3.91 dd (16.5, 7.0),
3.48 dd (16.5, 5.3)
7.24 br s, 6.97 br s
b
143.4 d
7.35 d (13.2)
5.87 dd (13.1, 10.2)
NH2
R1 Ile
NH
CO
a
a,b,c
Interchangeable.
Overlapping signals at 24.6, 24.7, 24.8, 24.9.
Overlapping signals at 25.0, 25.5, 25.8 ppm.
172.0 s
64.4 d
37.8 d
d
e
4.08 m
1.41 m
b
c
24.0 t
14.2 q, 10.5 q
1.41 m, 0.79 m
0.79 m,
c0
d
to interpret unambiguously a priori. With the planar structure in
hand it was possible to match the observed fragmentation patterns
with the sequence of amino acids deduced by NMR spectroscopy,
as exemplified by the MS spectra of callyaerin B (2), (see Fig. S9,
Supplementary data). No attempt was made to analyse such data
for the other compounds.
The stereochemistry of the various amino acid residues was
determined following acid hydrolysis of the parent peptide and
subsequent derivatization according to the advanced Marfey’s
method.14 Comparison of the resulting (N-(5-fluoro-2,4-dinitro-
0.42 t (7.3)
R2 Hyp
CO
a
b
c
d
173.3 s
55.6 d
37.7 t
68.7 d
56.7 t
4.26 m
2.08 m, 1.89 m
4.41 m
3.77 br d,
3.70 dd (11.4, 3.7)
5.36 d (3.1)
9.03 br s
OH
NH
CO
a
b
c
R3 Val
R4 Ile
172.0 s
66.1 d
27.1 t
19.2 q, 19.5 q
2.98 dd (10.7, 7.1)
2.65 m
0.83 d (6.6),
0.82 d (7.0)
8.81 br s
phenyl)-
appropriate standard amino acids using LC–MS indicated all amino
acids were of the -form.
L-leucinamide) amino acid derivatives with those of
L
NH
CO
a
An identical approach was used to establish the structures of
callyaerins A (1), B (2), C (3) and H (8). The fully assigned 1H and
13C NMR data are given in Tables 2–4 and 6 as well as a represen-
tative schematic of the sequence correlations in the ROESY and
HMBC spectra of callyaerin H (8) is shown in Figure 3. For the
remaining two compounds, callyaerins D (4) and F (6), there was
insufficient material available to record heteronuclear NMR spec-
tra. However, in both cases there were sufficient sequence correla-
tions in the ROESY spectra to afford a complete assignment of the
1H NMR data (Table 5), that unambiguously establish their struc-
tures as shown, for example, by the correlations of Figure 4 for cal-
lyaerin F (6). In all cases, the linear peptide chains of the callyaerins
originated with a proline attached to DAA via an amidic bond and
consequently showed a base peak in the ESI-MS through cleavage
of this bond to form a stable fragment of the cyclic peptide moiety.
This furnished immediate confirmatory evidence of the number
and nature of the amino acid residues in the side chain. In all cases
171.2 s
59.3 da
3.79 m
1.49 m
1.40 m, 1.13 m
0.81 d (6.9),
0.78 t (7.4)
7.44 d (7.0)
b
36.1 d
d
c
c0
d
15.2 q, 10.2 q
R5 Leu
NH
CO
a
172.3 s
49.1 db
4.61 m
1.77 m, 1.25 m
1.62 m
0.87 d (6.5),
0.86 d (6.6)
b
41.1 t
d
c
d, d0
21.0 qc, 21.0 qc
R6 Pro
R7 Pro
R8 Leu
CO
a
b
c
d
CO
a
b
c
d
171.4 s
64.0 d
4.05 m
26.2 t
2.30 m, 1.91 m
1.99 m, 1.91 m
3.61 m, 3.47 m
e
46.1 t
171.4 s
62.5 d
4.27 m
28.5 t
2.25 m, 1.61 m
1.94 m, 1.86 m
3.59 m, 3.31 m
6.72 d (10.0)
e
only L-amino acids were detected after acidic hydrolysis and deriv-
atization (see Table S2, Supplementary data).
The peptides are unusual in containing a considerable number
of proline (and in four cases c-hydroxyproline) residues, of which
47.0 t
NH
CO
a
172.4 s
50.0 db
4.61 m
one proline is always positioned at the beginning of the side chain,
while all others are found in the ring system. The remaining resi-
dues are predominantly hydrophobic amino acids such as, Ile,
Leu, and Phe. The basic structural unit of the callyaerins comprises
a cyclic peptide with a linear peptide side chain, both of variable
size, linked through a non-proteinogenic (Z)-2,3-diaminoacrylic
acid (DAA) functional group. Apart from our previous report of cal-
lyaerin G (7)12 there is only one other example of a compound con-
taining the DAA group from marine sources, namely callynormine
A isolated from the marine sponge C. abnormis.13 It was speculated
that DAA biogenetically arises from oxidation of a serine or cys-
teine unit to give a formyl glycine which will undergo Schiff base
formation with the amino group of the next amino acid in the ring,
followed by double bond migration.13 Thus such an arrangement of
a peptide fused with the DAA group affords a novel class of marine
natural products that are currently restricted to the sponge genus
Callyspongia. Interestingly, the DAA function is encountered in the
structures of antibiotics from actinobacteria, including capreomyc-
ins and viomycins from Streptomyces spp. as well as tuberactino-
mycins from Nocardia spp. which are used to treat tuberculosis.16
However, since these structures are otherwise only distantly
b
40.5 t
1.89 m, 1.57 m
1.65 m
0.96 d (6.6),
0.85 d (6.6)
d
c
d, d0
22.8 qc, 23.2 qc
C1 Pro
C2 Ile
CO
a
b
c
d
NH
CO
a
172.7 s
61.9 d
4.25 m
29.4 t
2.26 m, 1.50 m
1.83 m, 1.72 m
3.51 m, 3.21 m
7.47 m
e
48.7 t
171.1 s
58.6 da
3.79 m
1.67 m
1.17 m, 1.05 m
0.39 d (6.8), 0.70 t (7.3)
7.27 d (8.6)
b
34.9 d
d
c
c0
d
14.9 q, 11.0 q
C3 Phe
NH
CO
a
171.5 s
59.3 d
39.9 t
4.27 m
b
3.09 dd (13.9, 2.9),
2.63 dd (14.0, 12.1)
7.19–7.11 m
Others
C1: 138.0 s, C2,6:
129.0 d, C3,5:
127.7 d, C4: 126.1 d

S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
4951
Table 3
1H and 13C NMR data of callyaerin B (2) (DMSO-d6, 600 MHz)
Table 4
1H and 13C NMR data of callyaerin C (3) (DMSO-d6, 600 MHz)
A. A.
DAA
Position
dC
dH (J in Hz)
A. A.
DAA
Position
dC
dH (J in Hz)
NH
CO
a
8.28 br s
NH
CO
a
8.57 s
167.2 s
97.9 s
167.8 s
99.1 s
b
142.6 d
7.04 d (13.6)
b
143.3 d
7.65 d (13.7)
R1 Ile
R2 Hyp
R3 Ile
R4 Ile
R5 Leu
NH
CO
a
5.70 dd (13.6, 10.8)
R1 His
R2 Hyp
R3 Leu
R4 Leu
NH
CO
a
5.64 dd (13.7, 9.9)
172.1 s
64.6 d
37.9 d
172.3 s
59.7 d
34.3 t
2: 134.9 d,
4: 114.6 d
171.7 s
61.6 d
37.9 t
68.5 d
54.3 t
3.94 t (9.8)
1.45 m
4.48 ddd (10.5, 9.9, 4.0)
2.33 m, 2.20 m
NH1/3: 12.03 s, 2H: 7.39 s,
4H: 6.01 s
b
b
c
29.1 t
1.40 m, 1.00 m
Others
b
c0
d
0.85a, 0.76 t (7.5)
CO
a
b
c
173.4 s
59.2 d
37.5 t
68.7 d
56.7 t
CO
a
b
c
d
OH
NH
CO
a
b
c
4.24 m
2.10 m,1.89 m
4.40 br s
3.67 m, 3.80 m
5.40 d (2.4)
9.00 br s
4.14 dd (9.2, 4.3)
2.07 m, 1.64 m
4.06 m
3.54 m, 1.81 m
5.09 d (3.8)
d
OH
NH
CO
a
9.84 d (7.7)
172.1 s
64.2 d
32.4 d
172.5 s
52.2 d
3.07 m
2.55 m
1.40 m, 1.10 m
0.82a t, 0.78a
8.86 br s
4.16 ddd (11.4, 7.6, 3.9)
1.59 m, 1.51 m
1.73 m
0.86 d (6.6), 0.82 d (6.5)
7.57 d (6.3)
b
39.4 t
a
c
24.5 t
b
c0
d
d
20.7 q +b
NH
CO
a
NH
CO
a
b
c
d
CO
a
b
c
d
CO
a
b
c
171.3 s
57.8 d
36.2 d
169.9 s
49.2 d
3.81 (8.8)
1.55 m
4.42 ddd (9.1, 6.1, 5.1)
1.63 m, 1.22 m
1.47 m
b
39.7 t
a
c
25.5 t
1.45 m, 1.15 m
b
c0
d
0.84a, 0.81a
7.37 d (6.6)
t
b ꢂ 2
0.89 d (6.6), 0.89 d (6.5)
NH
CO
a
R5 Pro
R6 Pro
R7 Val
C1 Pro
C2 Leu
172.0 s
62.9 d
171.4 s
49.1 d
41.1 t
4.30 dd (9.0, 8.0)
2.34 m, 1.58 m
1.92 m
4.62 q (ꢁ6.7)
1.76 m, 1.30 m
1.60 m
29.2 t
a
b
c
24.7 d
47.2 t
172.0 s
63.9 d
3.67 m, 3.19 m
b
d, d0
CO
a
0.88a
R6 Pro
R7 Pro
R8 Leu
172.3 s
63.9 d
24.9 t
29.1 t
46.1 t
171.4 s
61.3 d
25.7 t
29.7 t
47.0 t
4.39 dd (11.4, 7.2)
2.20 m, 1.89 m
2.00 m
3.56 m
7.32 d (10.3)
4.03 t (8.2)
26.8 t
a
b
c
d
CO
a
b
c
d
2.30 m, 1.90 m
2.00 m, 1.90 m
3.60 m, 3.48 m
d
46.1 t
NH
CO
a
b
c
CO
a
b
c
171.8s
56.6 d
29.3 d
17.4 q, 18.9 q
173.7 s
62.3 d
4.25 m
4.67 dd (10.2, 4.4)
2.54 m
1.11 d (7.0), 0.97 d (6.9)
2.20 m, 1.82 m
1.90 m, 1.50 m
3.55 m, 3.35 m
6.63 (10.0)
NH
CO
a
4.32 dd (10.8, 7.0)
2.29 m, 1.53 m
1.88 m, 1.77 m
3.48 m, 3.28 m
7.90 d (6.2)
172.7 s
50.0 d
40.5 t
29.1 t
a
4.55 ddd (10.7, 10.4, 3.7)
1.70 (m), 1.60 (m)
1.47(m)
b
c
d
48.6 t
24.6 d
NH
CO
a
b
c
b
d, d0
CO
a
0.88a, 0.81a
171.9 s
52.6 d
C1 Pro
C2 Ile
171.2 s
62.5 d
26.2 t
29.7 t
48.6 t
3.90 ddd (11.1, 7.2, 3.8)
1.64 m, 1.16 m
1.64 m
0.87 d (6.5), 0.75 d (6.5)
7.80 d (9.3)
4.25 m
38.4 t
a
b
c
d
NH
CO
a
2.55 m, 1.80 m
1.70 m, 1.45 m
3.52 m, 3.15 m
7.58 d (10.4)
d
20.7 q + b
C3 Phe
C4 Gly
NH
CO
a
171.0 s
55.1 d
37.5 t
2,6: 129.6 d,
3,5: 127.9 d,
4: 126.4 d
172.1 s
57.8 d
36.4 d
4.24 ddd (12.1, 9.3, 3.1)
3.30 m, 2.98 dd (13.3, 12.0)
2,6: 7.35 m, 3,4,5: 7.15 m
4.10 dd (9.4, 9.2)
1.82 m
b
b
others
c
25.0 t
1.45 m, 1.15 m
b
c0
d
0.86a, 0.82a
7.23 d (9.5)
t
C3 Ile
NH
CO
a
NH
CO
a
7.74 dd (7.1, 5.1)
172.7 s
57.4 d
36.2 d
170.7 s
42.1 t
4.15 dd (9.5, 5.0)
1.90 m
1.25 m, 1.00 m
0.78a, 0.71 t (7.5)
7.17 s, 6.97 s
3.88 dd (17.0, 7.2),
3.54 dd (16.8, 5.0)
7.20 br s, 7.08 br s
b
c
23.5 t
NH2
b
c0
d
a
Interchangeable signals at 24.4 (x2), 24.5, 24.6, 25.5, 25.9.
Interchangeable Me signals at 21.4, 22.8, 23.0, 23.4.
NH2
b
a
Signal overlap prevents determination of couplings.
Specific assignments of the methyl signals are not possible. However the rela-
b
tive assignments are 23.1, 22.7, 22.1, 20.8 (d-Leu), 15.7 (ꢂ2), 15.3, 15.2, 14.4 (
c-Ile),
and 11.4, 11.0, 10.7, 10.3, 9.8 ppm (d-Ile).
Callyaerins A–F (1–6) and H (8) showed biological activity in
various assays. Their cytotoxicity activity (Table 7) was assessed
against the tumour cell line L5178Y (mouse lymphoma), and in
part against HeLa (human cervix carcinoma) and PC12 (rat brain
related, it does not seem justified to speculate on the microbial ori-
gins of the callyaerins.

4952
S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
H
N
O
O
H
N
O
H
H
N
H
H
N
H
H2N
H
O
O
N
O
H
N
NH
O
H
O
H
O
O
NH
H
H
N
NOE
N
H
HMBC
Figure 3. Schematic of the ROESY and HMBC correlations of H (8).
tumour), Callyaerins E (5) and H (8), in particular, exhibited strong
activity against the L5178Y cell line with ED50 values of 0.39 and
recorder Model 561, using 1 cm quartz cell. Optical rotations were
measured on a Perkin–Elmer Model 341 LC polarimeter. ESI- and
LC–MS spectra were obtained with a Thermofinnigan LCQ DECA
mass spectrometer coupled to an Agilent 1100 HPLC system
equipped with a photodiode array detector. HRESIMS were deter-
mined on a Micromass Q-Tof 2 mass spectrometer. Column chro-
matography was performed on Silica Gel 60 (0.04–0.063 mm,
0.48
previously.12 The remaining congeners were less active against the
L5178Y cell line with ED50 values ranging from 2.92 to 4.14 M,
lM, respectively, similar to those of callyaerin G (7) reported
l
apart from F which was inactive. Callyaerin E (5) was the most
active compound in the three cell lines tested, while callyaerin F
(6) showed the least activity. In addition, callyaerin A (1) exhibited
strong inhibitory properties towards C. albicans and moderate
activity against the gram negative Escherichia coli while it was only
mildly active or inactive against the gram positive Staphylococcus
aureus and Bacillus subtilis, respectively (Table 8). Callyaerin E (5)
displayed strong antimicrobial activity towards C. albicans and B.
subtilis but was only mildly active against the two remaining bac-
terial strains. Some general conclusions regarding a structure–
activity relationship (SAR) was apparent according to the bioassay
results, which indicated that increasing the number of proline res-
idues in the cyclic moiety enhanced the cytotoxicity, while replace-
ment of a proline with a hydroxyproline reduced the cytotoxicity.
Reduction of the cyclic moiety to a six-membered ring as in callya-
erin F caused a total loss of activity against all the tumour cell lines
tested. Proline-rich cyclic peptides have been described for their
medicinal properties, particularly their antimicrobial activity17a
while compounds of marine origin such as stylopeptide,17b wainu-
nuamide,17c axinellins,18 and phakellistatins19 have been reported
to be cytotoxic. Proline residues in these peptides have been linked
to conformational control in a molecule by providing rigidity due
to the restricted dihedral angles in proline which reduces other
conformation possibilities in a cyclic structure.20
Merck) and RP-18 Lobar columns (40–63 lm, 25 mm id
ꢀ310 mm, Merck, Darmstadt, Germany). Pre-coated Silica Gel 60
F254 plates (Merck) were used for TLC analyses.
1D (1H and 13C) and 2D (TOCSY, ROESY, HMBC and HMQC) nu-
clear magnetic resonance spectra were recorded on a Bruker
AVANCE DMX600 NMR spectrometer (apart from some 1D 13C
spectra on a Bruker ARX 400 instrument) using DMSO-d6 as sol-
vent and internal reference (1H: 2.50 ppm, 13C: 39.5 ppm). 1H
chemical shifts and coupling constants were evaluated from 1H
spectra after Gaussian multiplication. In cases of signal overlap
chemical shifts were evaluated from cross peaks in the 2D TOCSY
spectrum. 13C shifts were from the 1D spectrum or from the 2D
correlations.
HPLC separation was performed on a semi-preparative HPLC
system consisting of a Lachrom-Merck Hitachi L-7100 pump and
a L-7400 UV detector using a C-18 column (300 ꢂ 8 mm id, pre-
filled with Eurospher 100, Knauer, Berlin, Germany), with a flow
rate of 5.0 mL/min. Solvents were distilled prior to use and spectral
grade solvents were used for spectroscopic measurements.
4.2. Animal material
The sponges were collected by scuba diving at a depth of
4–5 m at Ambon in Indonesia in 1996. Freshly collected sponges
were frozen immediately after collection and then freeze-dried.
3. Conclusion
Investigation of the Indonesian Callyspongia aerisuza sponge
yielded novel proline-rich peptides, the callyaerins. The analogues
showed biological activity in a number of assays and indicated
their cytotoxicity increased with the number of proline residues
present in the molecule. This study adds considerably to our
knowledge of the secondary metabolites produced by the genus
Callyspongia which has been predominated until now by polyacet-
ylenic10 and nitrotetradecenyl pyridine11 metabolites.
A
voucher specimen has been deposited in the Zoological
Museum, University of Amsterdam, under the registration no.
ZMAPOR 17717.
4.3. Extraction and isolation
The sponge sample was freeze-dried (300 g) and was repeatedly
extracted with MeOH (2 L ꢂ 4). The total MeOH extract was evap-
orated under reduced pressure to dryness (310 mg) and subjected
to vacuum liquid chromatography (VLC) using hexane, CH2Cl2,
EtOAc, and MeOH. The preliminary screening for cytotoxic activity
of the different fractions revealed that the EtOAc fraction showed
strong activity with 100% mortality at concentrations of 20 and
4. Experimental
4.1. General experimental procedures
The UV spectra were determined using a Perkin–Elmer double
beam spectrophotometer Model 550 S, attached to a Hitachi
50 lg/mL in the brine shrimp test. The bioactive EtOAc fraction

S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
4953
Table 5
1H NMR data of callyaerin D (4) and F (6) (600 MHz, DMSO-d6)
Callyaerin D (4)
Callyaerin F (6)
dH (J in Hz)
8.25 s
A. A.
DAA
Position
dH (J in Hz)
Sequential NOEs
1-NH, 7
A. A.
DAA
Position
Sequential NOEs
NH
a
8.42 br s
a
NH
a
1-NH
b
7.38 d (8.8)
b
7.25 d (14.0)
R1 Ile
R2 Ile
NH
a
b
5.30 t (10.7)
4.41 m
1.98 m
DAA–NH
R1 Val
R2 Pro
R3 Val
R4 Phe
NH
a
b
c
a
b
c
d
NH
a
5.14 dd (10.1, 13.7)
4.38 br d (9.8)
2.31 m
0.87 d (6.9), 0.66 d (6.9)
3.95 dd (10.3, 6.7)
2.17 m, 1.81 m
2.03 m, 1.84 m
3.86 m, 3.74 m
7.54 d (5.1)
2-d
2-NH
2-NH
c
0.94 m, 1.20 m
c0
d
0.83a, 0.84a
3-NH
NH
a
b
7.64 d (4.9)
4.00 dd(4.9, 3.5)
1.81 m
1-b, 1-
c
3-NH
c
1.25 m, 1.30 m
c0
d
0.80a, 0.80a
3.86 m
2.11 m
4-NH
R3 Phe
R4 Pro
NH
a
b
Others
a
7.50 d (6.0)
4.61 m
3.06 dd (13.1, 9.7), 2.77 dd (13.1, 3.5)
7.25 d (8.8), 7.23 d (6.9), 7.21 d (7.6)
4.17 t (7.1)
2-
a
, 4-d
, 4-d
b
c
NH
a
4-a
0.93 d (6.9), 0.86 d (6.9)
7.50 d (6.1)
4.54 ddd (9.3, 6.1, 3.5)
3.01 dd (12.9, 9.7)
2.71 dd (12.8, 3.1)
7.14–7.28 m
5-d
5-d
3-a
b
b
c
d
a
b
c
d
a
b
c
d
NH
a
b
c
d
a
b
c
d
NH
a
b
1.40 m,1.70 m
1.42 m,1.66 m
2.23 m, 3.27 m
4.40 m
2.00 m, 2.21 m
1.91 m
3.65 m, 3.93 t (6.3)
4.30 t (8.6)
1.75 m, 2.17 m
1.60 m, 1.70 m
3.30 m, 3.53 t (8.8)
7.54 d (9.5)
4.47 td (9.4, 5.5)
1.85 m, 1.71 m
1.70 m
0.80 m, 0.82 m
4.32 m
1.70 m, 2.16 m
1.80 m, 1.90 m
3.61 m, 3.38 m
7.76 d (6.3)
others
a
b
c
d
a
b
c
d
NH
a
b
c
d
NH
a
R5 Pro
C1 Pro
C2 Leu
4.28 m
DAA–NH
3-NH
2.09 m, 1.80 m
1.91 m, 1.83 m
3.58 m, 3.35 m
4.26 m
2.19 m, 1.42 m
1.73 m, 1.45 m
3.26 m, 2.31 m
7.66 d (9.6)
4.62 td (10.1, 3.9)
1.93 m, 1.67 m
1.72 m
0.94 d (6.9), 0.84 d (6.9)
6.17 d (7.8)
4.42 td (7.6, 4.5)
2.93 m
7.14–7.28 m
7.52 d (8.5)
3.98 dd (8.3, 6.1)
1.75 m
R5 Hyp
R6 Pro
R7 Leu
2-NH
3-NH
DAA–NH
DAA–NH
C3 Phe
C4 Ile
4-NH
C1 Pro
C2 Ile
2-a, 2-NH
b
others
NH
a
2-
1-
a, 2-NH
a
Terminal-NH2
b
3.93 t (6.3)
1.90 m
3-NH
c
1.29 m, 1.10 m,
0.76 d (6.8), 0.77 t (7.3)
7.04 br s, 6.95 br s
c0
d
c
1.40 m, 1.28 m
NH2
c0
d
0.80a, 0.85a
C3 Asn
NH
7.88 d (7.6)
2-
a
a
b
NH2
NH
a
4.55 q (ꢁ7)
4-NH
2.69 dd (15.1, 6.9), 2.35 dt (15.1, 6.9)
6.93 s, 7.31 s
7.57 d (6.9)
C4 Ala
C5 Ile
3-a
5-NH
4.16 m
b
NH
a
1.25 d (6.9)
7.34 d (6.7)
4.05 dd (8.9, 6.7)
1.77 m
4-b
Terminal-NH2
b
c
1.12 m, 1.45 m
c0
d
0.85a, 0.87a
a
Signal overlap prevents determination of couplings.
(120 mg) was chromatographed over a silica gel column using a
CH2Cl2/MeOH gradient to give five fractions. Fraction-1 (30 mg)
was submitted to semi-preparative HPLC (HPLC gradient program:
60:40 MeOH/H2O at 0 and 5 min; 100:0 MeOH/H2O at 38 and
and 5 min; 100:0 MeOH/H2O at 38 and 45 min and a flow rate
5.0 mL/min) to afford callyaerin F (2 mg). Fraction-4 was purified
over a silica gel column using CH2Cl2/MeOH (85:15) and then by
semi-preparative HPLC (HPLC gradient program: 60:40 MeOH/
H2O at 0 and 5 min; to 100 MeOH at 38 min, held until 45 min with
a flow rate of 5.0 mL/min) to yield callyaerins A (6 mg) and B
(8 mg). Fraction-5 was subjected to RP-18 reversed phase column
chromatography using MeOH/H2O (85:15) to afford callyaerin D
(3 mg).
45 min and
a flow rate 5.0 mL/min) to yield callyaerins C
(5.5 mg) and E (7.5 mg). Fraction-2 was subjected to RP-18 re-
versed phase column chromatography using a MeOH/H2O gradient
to give callyaerin H (6 mg). Fraction-3 was purified via semi-pre-
parative HPLC (HPLC gradient program: 60:40 MeOH/H2O at 0

4954
S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) data,
see Table 3.
Table 6
1H and 13C NMR NMR data of callyaerin H (8) (600 MHz, DMSO-d6)
A.A.
Position
dC
dH (J in Hz)
4.3.3. Callyaerin C (3)
DAA
NH
CO
2
8.47 br s
White amorphous powder, [
a
]
ꢀ52 (c 0.15, MeOH). UV kmax
167.2 s
98.8 s
143.3 d
D
(MeOH) 208 and 283 nm. (+) ESI-MS (rel. int.%): m/z 1268.5
[M+H]+ (100), 1290.7 [M+Na]+ (45). HRESI-TOFMS: m/z 1268.718
(calcd for C63H94N15O13, 1268.7156). 1H NMR (600 MHz, DMSO-
d6) and 13C NMR (150 MHz, DMSO-d6) data, see Table 4.
3
7.23 d
5.26 dd (13.7, 10.0)
R1 Val
R2 Pro
R3 Val
R4 Phe
NH
CO
a
172.4 s
62.7 d
29.3 d
15.6 q, 21.6 q
172.4 s
59.8 d
26.8 t
24.3 t
46.2 d
4.42 dd (10.0, 1.4)
2.35 m
0.68 d (6.7), 0.89 d (6.9)
b
cc0
CO
a
4.3.4. Callyaerin D (4)
3.97 dd (10.5, 6.8)
2.03 m, 1.82 m
2.18 m, 1.82 m
3.82 m, 3.75 m
7.59 d (5.1)
White amorphous powder, [
a
]
ꢀ49 (c 0.2, MeOH). UV kmax
D
b
c
d
NH
CO
a
(MeOH) 205 and 285 nm. (+) ESI-MS (rel. int.%): m/z 1386.5
[M+H]+ (100), 1408.8 [M+Na]+ (90), 861 (35) [Mꢀ(Ile+Ala+Asn+Ile+
Pro+NH2)]+; FAB-MS+ 861.5. HRESI-TOFMS: m/z 1386.817 (calcd for
69H108N15O15, 1386.8071). 1H NMR (600 MHz, DMSO-d6) data, see
170.7 s
61.0 d
28.8 d
18.4 q,18.5 q
C
3.86 t (4.7)
2.10 m
0.93 d (7.0), 0.86 d
7.51 d (5.8)
Table 5.
b
cc0
NH
CO
a
4.3.5. Callyaerin E (5)
168.9 s
53.5 d
36.2 t
White amorphous powder, [
a
]
D
ꢀ68 (c 0.25, MeOH). UV kmax
4.57ddd (9.6, 5.9, 3.8)
3.03 dd (13.1, 9.7),
2.77 dd (13.1, 3.6)
7.2–7.3 m
b
(MeOH): 206 and 282 nm. (+) ESI-MS (rel. int.%): m/z 1262.5
[M+H]+ (95), 1284.7 [M+Na]+ (90), 865 [Mꢀ(Pro+2Ile+Gly+NH2)]+
(100). HRESI-TOFMS: m/z 1284.710 (calcd for C66H95N13O12Na,
1284.71209), 1300.6880 (calcd for C66H95N13O12K, 1300.6860). 1H
NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) data,
see Table 1.
others
1: 137.9 s,
2,6: 128.9 d,
3,5: 128.1 d,
4: 126.5 d
171.5 s
63.2 d
29.5 t
24.3 t
46.4 d
171.3 s
63.6 d
R5 Pro
R6 Pro
R7 Leu
CO
a
b
c
d
CO
a
b
c
d
4.34 dd (11.4, 7.5)
1.80–2.10 m
1.80–2.10 m
4.3.6. Callyaerin F (6)
3.63 m, 3.35 m
White amorphous powder, [
a
]
D
ꢀ32 (c 0.15, MeOH). UV kmax
(MeOH): 204 and 279 nm. (+) ESI-MS (rel. int.%): m/z 548.3, 1095
[M+H]+ (75), 1117 [M+Na]+ (100). (ꢀ) ESI-MS m/z (rel. int.%):
1093 [MꢀH]ꢀ (40) and 1140 [M+HCOOH]ꢀ (100). HRESI-TOFMS:
m/z 1094.6380 (calcd for C58H84N11O10, 1094.6324), 1116.620
4.17 dd (9.3, 7.7)
2.21 m, 1.69 m
1.45 m
3.27 m
7.54 d (9.6)
29.5 t
25.5 t
46.5 d
NH
CO
a
(calcd for
C
58H83N11O10Na, 1116.6222). 1H NMR (600 MHz,
172.2 s
50.4 d
41.0 t
DMSO-d6) data, see Table 5.
4.51 ddd (9.8, 9.8, 4.3)
1.77 m
b
c
24.7 d
1.85 m
0.86 d, 0.82 d
d, d0
CO
a
19.9 q, 22.6 q
171.9 s
63.6 d
29.5 t
25.6 t
4.3.7. Callyaerin H (8)
C1 Pro
C2 Ile
White amorphous powder, [
a
]
ꢀ93 (c 0.3, MeOH). UV kmax
D
4.34 dd (9.6, 7.8)
2.16 m, 1.72 m
1.81 m, 1.55 m
3.48 br t (8.0), 3.24 m
7.50 d (9.1)
(MeOH): 208 and 284 nm. (+) ESIMS (rel. int.%): m/z 1044 [M+H]+
(75), 1066 [M+Na]+ (100), and 817 [Mꢀ(Pro+Ile+NH2)]+ (90). HRES-
I-TOFMS: m/z 1066.608 (calcd for C54H81N11O10Na, 1066.6066),
b
c
d
NH
CO
a
48.5 d
1082.581 (calcd for
C
54H81N11O10K, 1082.5805). 1H NMR
173.2 s
57.2 d
35.5 d
(600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) data, see
4.01 dd (9.2, 6.1)
1.92 m
Table 6.
b
c
28.6 t
15.3 q, 11.3 q
1.21 m, 1.40 m
0.85 d (6.6), 0.82 t (7.3)
6.96 br s, 6.89 br s
c0
d
NH2
4.4. Acid hydrolysis and absolute configuration of component
amino acids using LC–MS analysis of the Marfey derivatives14
Each of the isolated peptides (1.0 mg) was treated separately
with 2 mL 6 N HCl (pa) and heated at 110 °C for 24 h in ampoules
previously sealed under N2 gas. The resulting solutions were con-
centrated, with consecutive addition of H2O (5 mL each) to ensure
4.3.1. Callyaerin A (1)
White amorphous powder; [
(MeOH) 207 and 281 nm. (+) ESI-MS (rel. int.%): m/z 1357.6
[M+H]+ (80), 1379.8 [M+Na]+ (50). HRESI-TOFMS: m/z 1357.821
(calcd for C69H109N14O14, 1357.8248). 1H NMR (600 MHz, DMSO-
d6) and 13C NMR (150 MHz, DMSO-d6) data, see Table 2.
a]
ꢀ80 (c 0.12, MeOH); UV kmax
D
complete elimination of HCl. To 50
authentic amino acid standard at comparable concentration),
100 L FDNPL (1% N-(5-flouro-2,4-dinitrophenyl)- -leucinamide
in acetone) and 20 L 1 M NaHCO3 were added. The mixture was
heated over a hot plate at 40 °C for 1 h with frequent mixing. After
cooling, 10 L of 2 M HCl was added and then concentrated to dry-
ness before dissolving in 1000 L MeOH. and amino acid stan-
lL of each acid hydrolysate (or
l
L
l
4.3.2. Callyaerin B (2)
Yellowish-white amorphous powder, [
a]
D
ꢀ89 (c 0.2, MeOH).
l
UV kmax (MeOH) 210 and 284 nm. (+) ESI-MS (rel. int.%): m/z
1280.6 [M+H]+ (15), 1302.8 [M+Na]+ (100); FAB-MS+ 940.2. HRES-
I-TOFMS: m/z 1280.839 (calcd for C65H110N13O13, 1280.8268). 1H
l
L
D
dards were treated separately with FDNPL in the same manner.
The FDNPL derivatives were analysed using LC–MS by comparison

S. R. M. Ibrahim et al. / Bioorg. Med. Chem. 18 (2010) 4947–4956
4955
O
O
O
N
H
H
N
O
H
H
N
H
HN
H
N
H
H2N
O
H
NH
H
O
N
O
H
H
O
H
NH
O
N
H
O
H
NH
H
NOE
TOCSY
Figure 4. Schematic of the TOCSY and ROESY sequential correlations of callyaerin F (6).
Table 7
Cytotoxicity test of the isolated callyaerins
Compound
Brine shrimp assay mortality (%)
g/mL 50 g/mL
% Growth inhibition ED50
Hela cell
(lM)
20
l
l
L5178Y
PC12 cell
Total extract
Ethyl acetate extract
Hexane extract
Callyaerin A
Callyaerin B
Callyaerin C
Callyaerin D
Callyaerin E
100
95
30
n.t.
15
n.t.
n.t.
45
100
100
50
n.t.
35
n.t.
n.t.
70
n.t.
n.t.
n.t.
3.61
4.14
2.92
3.03
0.39
>9
n.t.
n.t.
n.t.
n.t.
>8
n.t.
n.t.
3.4
>9
n.t.
n.t.
n.t.
n.t.
>8
n.t.
n.t.
3.8
>9
Callyaerin F
n.t.
n.t.
Callyaerin Ga
Callyaerin H
0.41
0.48
4.43
n.t.
>8
n.t.
30
55
n.t. not tested.
a
From our previous work.12
Germany) for cytotoxicity testing. This project was supported by
grants of the BMBF and MOST awarded to Professor Peter Proksch
and Professor Wenhan Lin.
Table 8
Antibiotic assay of the isolated callyaerins
Compound
Zone of inhibition (mm)
SA 5–10
lL
BS 5–10
lL
EC 5–10
lL
CA 5–10 lL
Supplementary data
Callyaerin A
Callyaerin B
Callyaerin C
Callyaerin D
Callyaerin E
Callyaerin F
Callyaerin H
9–9
11–11
0–0
0–0
9–10
0–7
0–0
0–0
7–10
12–12
15–17
0–9
10–15
7–10
0–0
0–0
9–11
0–0
25–30
15–15
0–0
0–7
20–20
0–0
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.012.
Reference and notes
n.t.
n.t.
n.t.
n.t.
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