New cyclic peptides from the Indonesian sponge Theonella swinhoei

Article abstract of DOI:10.1016/S0040-4020(00)00762-6

Three new cyclic peptides, barangamides B, C, and D and a new depsipeptide, theonellapeptolide IIe as well as known theonellapeptolides Ia, Id, Ie, IId have been isolated from the sponge Theonella swinhoei collected in Baranglompo Island, Indonesia The structures of barangamides were elucidated by interpretation of NMR data and application of Marfey's method. The structure of theonellapeptolide IIe, a mixture of several conformers showing complex NMR spectra, was determined by MS analysis of a ring-opened product and by chemical reaction. Some of the theonellapeptolides showed mild immunosuppressive activity, while barangamide A was inactive. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00762-6

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9079±9092
New Cyclic Peptides from the Indonesian Sponge
Theonella swinhoei
Michael C. Roy,a Ikuko I. Ohtani,a Toshio Ichiba,b Junichi Tanaka,a Rachmaniar Sataric and
Tatsuo Higaa,
*
aDepartment of Chemistry, Biology, and Marine Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan
bOkinawa Industrial Technology Center, 12-2 Suzaki, Gushikawa, Okinawa 904-2234, Japan
cResearch and Development Centre for Oceanology, LIPI, JL. Pasir Putih I, Ancol Timur, Jakarta 11048, Indonesia
Dedicated to Professor Emeritus Paul J. Scheuer on the occasion of his 85th birthday, and to mark his 50th year of active research at University of Hawaii
Received 8 May 2000; accepted 19 July 2000
AbstractÐThree new cyclic peptides, barangamides B, C, and D and a new depsipeptide, theonellapeptolide IIe as well as known
theonellapeptolides Ia, Id, Ie, IId have been isolated from the sponge Theonella swinhoei collected in Baranglompo Island, Indonesia.
The structures of barangamides were elucidated by interpretation of NMR data and application of Marfey's method. The structure of
theonellapeptolide IIe, a mixture of several conformers showing complex NMR spectra, was determined by MS analysis of a ring-opened
product and by chemical reaction. Some of the theonellapeptolides showed mild immunosuppressive activity, while barangamide A was
inactive. q 2000 Elsevier Science Ltd. All rights reserved.
Numerous interesting compounds have been reported from
the sponge Theonella swinhoei belonging to the Order
Lithistida. Examples include swinholide A, a potent cyto-
toxic macrolide,1 cyclotheonamide A, a cyclic pentapeptide
possessing potent inhibitory activity against thrombin, tryp-
sin and plasmin,2,3 and other acyclic and cyclic polypeptides
ranging from cyclic depsipeptide to large bicyclic peptides.4
(2), and a new cyclic peptide, barangamide A (3).11 Further
investigation of this sponge resulted in the isolation of three
new cyclic peptides, barangamides B (4), C (5), and D (6)
and four new depsipeptides, theonellapeptolides IIe (7), IV,
V, and VI, as well as known theonellapeptolides Ia (8) and
Theonellapeptolides (Ia±e5,6 and IId7), tridecapeptide
lactones, have been reported from an Okinawan specimen
of T. swinhoei. These peptides are characterized by the
presence of high proportions of d-amino acids, N-methyl
amino acids, and b-amino acids. Biological activity of
these compounds includes cytotoxicity, ion-transport
activity for Na1 and K1 ions,6 and Na1, K1-ATPase
inhibitory activity.8 Other theonellapeptolides (III series)
exhibiting cytotoxicity have also been reported from a
New Zealand deep water sponge, Lamellomorpha
strongylata, belonging to a different Order.9
In our screening for cytotoxicity of over 300 marine
organisms collected in Indonesia, a lipophilic extract
(MeOH) of the sponge T. swinhoei10 was shown to be active
against KB cells. We have recently reported the isolation of
two known depsipeptides, theonellapeptolides Id (1) and IId
Keywords: cyclic peptide; depsipeptides; sponge; immunosuppressive
activity.
* Corresponding author. Tel.: 181-98-895-8558; fax: 181-98-895-8538;
e-mail: thiga@sci.u-ryukyu.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00762-6

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M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
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9082
M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
d
d

M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
9083
Ie (9). Since these compounds have some common struc-
tural features with those of well known immunosuppressive
drugs, the cyclosporins, we are interested to see whether
they have immunomodulatory activity and, if so, their
structure±activity relationship. In this paper we now report
the isolation and structure elucidation of 4±7 and a
preliminary result on the screening of immunosuppressive
activity of 1±3 and 8.
The sponge (600 g dry weight) collected at Baranglompo
Island, Indonesia by SCUBA was dried in the sun, and
extracted successively with heptane, ethyl acetate, acetone,
and methanol. The concentrated acetone extract (14.6 g)
was subjected to separation on silica gel (CH2Cl2±MeOH)
and ODS (MeOH) and by HPLC on ODS (MeCN±MeOH±
H2O, MeOH±H2O, MeCN±MeOH±H2O±TFA) to afford
barangamides A (3, 11 mg), B (4, 1.3 mg), C (5, 0.8 mg),
and D (6, 1.8 mg) and theonellapeptolides IIe (7, 12 mg), V
(7 mg), and VI (5 mg) along with known theonellapepto-
lides Id (1), IId (2), and Ia (8). The ethyl acetate extract
Figure 1. HOHAHA (bold line), ROESY (dashed arrow) and HMBC
correlations (arrow) for barangamide C (5).
Figure 2. ROESY and NOEDS correlations and assignment of isomeric isoleucine units in 3 and 5.

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M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092

M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
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d
d

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M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
(5.9 g) was separated by a silica gel (CH2Cl2±MeOH)
column followed by HPLC on ODS (MeCN±MeOH±
H2O, MeCN±MeOH±H2O±TFA) to afford theonellapepto-
lides IV (9 mg), V (24 mg), and VI (5.5 mg) along with 1±9.
Swinholide A (11.3 mg)1 was also isolated from the ethyl
acetate extract as a cytotoxic principle.
the allo-Ile unit (C28±33) as shown in B. The other unit
(C37±42) was assigned as Me-Ile by NOE between 38-NMe
and H2-40 and by no NOE between 38-NMe and H3-42 (C).
The validity of this method was con®rmed by reaching the
same conclusion regarding the position of d-allo-Ile and
d-allo-Me-Ile units in 1, the structure of which has been
determined by a more elaborate method.5 The two isomeric
Me-Ile units, (C5±10, C37±42) of 5 were assigned in the
same manner (Fig. 2) to complete the total structure as
shown. Barangamide C (5) differs from 3 only in the substi-
tution of a d-Val for a d-allo-Ile. Barangamide C (5) is a
cyclic undecapeptide possessing the same amino acid
sequence with the ring portion of the known theonellapep-
tolide Ia (8), which was also isolated in this study. Similar
relationship is found between barangamide A (3) and
theonellapeptolide Id (1).
The molecular formula of barangamide C (5) was deter-
mined as C53H95N11O12 by high-resolution FABMS (MH1
1078.7230, D 21.0 mmu), which was 14 mass units (CH2)
1
smaller than that of barangamide A (3). The H and 13C
NMR spectra of 5, which were quite similar to those of 3,
contained signals for eight NH (dH 6.3±9.2), three NMe (dH
2.64, 2.97, 3.29) and eleven CyO (dC 169±176) (Table 1).
Cyclic nature was suggested by negative ninhydrin reaction.
NMR analyses indicated the presence of eleven amino acid
residues, b-Ala (£3), Ala, Me-Ile (£2), Leu (£2), Thr, Val,
and Me-Val, the same composition found in 3 except for one
residue, i.e. Val (allo-Ile in 3). One of the Me-Ile was the
Both barangamides B (4) and D (6) had the same molecular
formula, C53H95N11O12, as determined by their high-
1
1
allo-form as shown by comparison of the H NMR data of
resolution FABMS data. The H and 13C NMR spectra of
the acid hydrolysate of 5 with those of authentic samples.
Interpretation of 2D NMR including HOHAHA, HMBC and
NOEDS data (Table 1) led to the planar structure of 5
(Fig. 1). Application of the Marfey's method12 enabled us
to determine the chirality of the residues as l-Ala, l-Me-Ile,
d-allo-Me-Ile, d-Leu (£2), l-Thr, d-Val and l-Me-Val.
4 and 6 were similar to those of 5 (Table 2). Since only small
amounts of samples were isolated, structure elucidation of 4
and 6 was mainly carried out by the analysis of HOHAHA
and ROESY data. Interpretation of HOHAHA spectrum
indicated the structures of the component amino acids
(Fig. 3), which were con®rmed by comparison of 1H
NMR spectrum of their acid hydrolysates with authentic
samples. The absolute con®guration of each amino acid
was determined by the Marfey analysis. Detailed analysis
of ROESY and some HMBC data led to the structures of
4 and 6 as shown in Fig. 3. Barangamides B (4) and D (6)
are closely related to theonellapeptolides Ib and Ic,
respectively.
As observed in 3, 5 also had two isomeric Me-Ile units
(C5±10, C37±42). In general, it is dif®cult to distinguish
diastereomers by NMR. We have successfully determined
the positions of the two isomeric Me-Ile units in 3 by exami-
nation of coupling constants and NOE data (Fig. 2).11 The
essence of this method is as follows. Authentic samples of
isoleucine and allo-isoleucine showed small vicinal
coupling constants (4 Hz) for H-2 and H-3, indicating no
rotation barrier around C2±C3 axis. On the other hand 3
showed large values for the corresponding coupling
constants (JH6,H7ˆ10.5 Hz and JH38,H39ˆ10 Hz) for the
Me-Ile units, suggesting restricted rotation and anti relation-
ship of these protons. In ROESY and NOEDS experiments,
the 6-NMe showed signi®cant NOE with H3-10 and no NOE
with H2-8 and H3-9, suggesting that this unit (C5±10) is the
allo-Me-Ile (A). Similar NOE relationship was observed for
We have isolated four known [Id (1), IId (2), Ia (8), Ie (9)]
and four new theonellapeptolides [IIe (7), IV, V, VI] in this
study. Each of these peptides existed as a mixture of several
conformers in such NMR solvents as CDCl3, CD3OD,
acetone-d6, CD3CN, C6D6, DMSO-d6, pyridine-d5, and
showed broad and complex signals. In CDCl3, 1 and 8
existed as a single major conformer, while in other solvents
their spectra were complex due to the existence of con-
formers. For example, 1 exhibited ®ve NMe signals in
Figure 3. HOHAHA (bold line), HMBC (!), and ROESY correlations ($) for barangamides B (4, a) and D (6, b).

M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
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M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
Figure 5. Methylation of theonellapeptolides (1, 2, 7, and 9) to 10.
CDCl3 and more than 7 NMe signals in DMSO-d6. The
structure of 1 could be con®rmed by the complete assign-
ment of NMR signals in CDCl3 (Table 1), while the NMR
analyses of other theonellapeptolides were quite dif®cult.
This dif®culty was overcome by analysis of MS fragment
ions of the ring-opened products. The ring open products 1a
and 1b were prepared from 1 by treating with NaOMe and
KOH, respectively. The negative FABMS showed weak
fragment ions, whereas the positive FAB, APCI, and/or
ESI MS of 1a and 1b showed expected fragment ions
(amide-bond cleavage), which were clearly assigned by
comparison of MS data of 1a and 1b (Fig. 4). The amino
acid sequences of other known theonellapeptolides (2, 8, 9)
were also deduced from the detailed analyses of the
fragment ions of the ring-opened products (2a,b, 8a,b,
9a,b). Thus, the amino acid sequences of theonellapepto-
lides can be deduced from the MS analyses of methanolysis
and hydrolysis products. Detailed analysis of MS data for 7a
and 7b, derived from the new theonellapeptolide 7,
suggested the sequence as shown in Fig. 4. The suggested
structure of 7 was an analogue having an l-Ala instead of
l-Me-Ala in the known theonellapeptolide Ie (9). The
relationship between 7 and 9 is the same as that found for
theonellapeptolides Id (1) and IId (2), thus we named 7
theonellapeptolide IIe. In the analysis of fragment ions of
7a and 7b, it was impossible to distinguish the amino acids
having the same molecular weights, such as Ala and b-Ala;
Leu, allo-Ile, and Me-Val; Me-Leu, Me-Ile, and Me-allo-
Ile. Therefore, validity of the suggested structure should be
con®rmed by other means. Methylation of 1, 2, 7, and 9 with
MeI and Ag2O gave the same compound 10, C78H141N13O16,
whose structure was con®rmed by FABMS (MH1 m/z
1517), Marfey's analysis, and 1H NMR analysis of
complete acid hydrolysate (Fig. 5). Thus, the structure of
theonellapeptolide IIe (7) was unambiguously established as
shown. The structures of other new theonellapeptolides IV,
V, VI are now under investigation and will be reported
elsewhere.
Theonellapeptolides and barangamides are tridecapeptide
Figure 6. Result of mixed lymphocyte reaction assay for theonellapeptolides Id (1), IId (2), Ia (8), and barangamide A (3). This ®gure was supplied by Dr Y.
Koezuka, Kirin Brewery Co. Ltd.

M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
9089
lactones and cyclic undecapeptides, respectively, posses-
sing a high proportion of N-methylated amino acids and
no aromatic amino acids. Similar structural features are
found in the cyclosporins, cyclic undecapeptides, some of
which are widely used as immunosuppressants after organ
transplant surgery.13 In order to test immunomodulatory
activity of these compounds we submitted samples of
theonellapeptolides (1, 2, 8) and barangamide A (3) for
mixed lymphocyte reaction (MLR) assay. The results are
shown in Fig. 6. Barangamide A (3) showed no activity
even at the highest concentration of 100 mg/mL, while all
theonellapeptolides (1, 2, 8) were active. Among the three
compounds, 2 showed the strongest immunosuppressive
activity. It is interesting to note that small variation in the
structures of these compounds signi®cantly affect their
biological activity. In a preliminary assay, 3 and 7 had no
cytotoxicity against L1210 tumor cells even at 10 mg/mL,
while theonellapeptolides Ia±Ie have been reported to be
cytotoxic (IC50 1.3±2.4 mg/mL) against the same cell
line.6 The observed immunosuppressive activity of
theonellapeptolides may largely originate from their cyto-
toxic effect. Further chemical and biological studies are
under way.
on ODS using MeOH±H2O (88:12), MeOH±MeCN±H2O
(8:2:0.8), and/or MeOH±MeCN±H2O (8:1:1) with 0.02%
TFA as eluents. The 2nd fraction yielded barangamides A
(3), B (4), C (5), and D (6) on repeated puri®cation on ODS
HPLC eluting with MeOH±H2O (88:12) and MeOH±
MeCN±H2O (3.5:3:3.5) with 0.1% TFA. The last fraction
gave theonellapeptolides Id (1) and Ia (8). Theonellapepto-
lides IId (2) and V were isolated from the 5th fraction. The
7th fraction yielded theonellapeptolides VI and IIe (7).
Similarly, ethyl acetate extract was ®rst chromatographed
on silica gel eluting with CH2Cl2 and MeOH (0±10%) to
give 16 fractions. The 13th fraction yielded 1±8 on further
puri®cation by HPLC. The 14th and 15th fractions were
puri®ed by ODS HPLC using MeOH±MeCN±H2O
(8:2:0.8) to yield 1, 2, and 7±9. The 10th fraction was
rechromatographed on silica gel using CH2Cl2 and
MeOH (0±10%) to give 8 fractions. Swinholide A was
isolated from the 4th and 5th fractions by PTLC on
silica gel (CH2Cl2±MeOH, 12:1). The 6th fraction yielded
theonellapeptolides IId (2), IIe (7), IV, V, and VI by
repeated reversed phase HPLC using MeOH±MeCN±H2O
(8:1:1) with 0.1% TFA. Theonellapeptolides Id (1) and Ia
(8) were also obtained from the 7th and 8th fractions by
repeated ODS HPLC, using MeOH±MeCN±H2O (8:1:1)
as eluent. The yields of 1±9 were 2000, 85, 22, 1.3, 0.8,
1.8, 12, 300, and 32 mg, respectively.
Experimental
1
Swinholide A. H and 13C NMR (CDCl3) were identical
General experimental procedures
with those reported.1
1
The H and 13C NMR spectra were recorded on a JEOL
a-500 spectrometer. The APCI, ESI, and FAB mass spectra
were measured on a JMS-700. The IR spectra and the
optical rotations were measured using a JASCO FT/IR-
Theonellapeptolide Id (1). A white crystalline solid; IR
(®lm) nmax 3315, 2962, 1735, 1652, 1627, 1542 cm21
;
LRFABMS, positive ion, m/z 1405 [M1H]1, negative ion,
1403 [M2H]2; 1H and 13C NMR (CDCl3) are listed in Table
1 (complete NMR data have not been described in the
300 and
a JASCO DIP-1000, respectively. High-
performance liquid chromatography (HPLC) was performed
on a Hitachi L-6000 pump equipped with a Hitachi RI
monitor (655A-30) and a Hitachi L-4000 UV detector,
using COSMOSIL packed ODS HPLC column (5C18-AR-
II, 10£250 mm). Kieselgel 60 (70±230 mesh, MERCK) and
Cosmosil 75C18-OPN (Nacalai Tesque) were used for
column chromatography. All solvents used were reagent
grade.
literature5). It was identi®ed by the H NMR comparison
1
with an authentic sample.
Theonellapeptolide IId (2). A white crystalline solid; IR
;
(®lm) nmax 3311, 2962, 1735, 1633, 1538 cm21
LRFABMS, negative ion, m/z 1389 [M2H]2. It was
identi®ed by the H NMR comparison with an authentic
sample.
1
Extraction and isolation
Barangamide A (3). A glassy solid (22 mg); [a]2D5ˆ238.48
A sample of T. swinhoei was collected by hand using
SCUBA in Baranglompo Island, Indonesia, in August,
1997, and sun dried prior to transportation to Okinawa.
The sample (600 g dry weight) was extracted successively
with heptane, ethyl acetate, acetone, and methanol. The
extracts were concentrated under reduced pressure to afford
1.5, 5.9, 14.6 and 51.6 g of materials, respectively. The ethyl
acetate and acetone extracts showed strong and moderate
cytotoxicity against L1210 cell line, respectively, while the
heptane and methanol extracts showed no activity at 10 mg/
mL. The acetone extract was chromatographed on silica gel
eluting with CH2Cl2 and increasing amount of MeOH
(1±100%) to give eight fractions. The 5th fraction was
further chromatographed on ODS using MeOH as eluent.
The fractions 1,3 were combined and subjected to further
separation by reversed phase HPLC on ODS using MeCN±
MeOH±H2O (16:3:1) to yield eight fractions. Final puri-
®cation of the peptides was carried out by repeated HPLC
(cˆ0.3, MeOH),11 [a]D28ˆ144.68 (cˆ0.3, CHCl3).
Barangamide B (4). A glassy solid (1.3 mg); [a]2D9ˆ176.58
(cˆ0.04, CHCl3); IR (®lm) nmax 3264, 2962, 1658,
1623 cm21
;
LRFABMS, negative ion, m/z 1076.7
[M2H]2, positive ion, m/z 1078.7 [M1H]1; HRFABMS
m/z 1078.7238 [M1H]1, calcd for C53H96N11O12
1078.7240 (D 20.2 mmu); H and 13C NMR (CDCl3) are
listed in Table 2.
1
Barangamide C (5). A glassy solid (0.8 mg); [a]2D6ˆ
157.88 (cˆ0.07, CHCl3); IR (®lm) nmax 3266, 2962,
1658, 1625 cm21; LRFABMS, negative ion, m/z 1076.7
[M2H]2, positive ion, m/z 1078.7 [M1H]1; HRFABMS
m/z 1078.7230 [M1H]1, calcd for C53H96N11O12
1
1078.7240 (D 21.0 mmu); H and 13C NMR (CDCl3) are
listed in Table 1.

9090
M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
Barangamide D (6). A glassy solid (1.8 mg); [a]2D9ˆ
184.68 (cˆ0.1, CHCl3); IR (®lm) nmax 3266, 2962, 1658,
1625 cm21; LRFABMS, negative ion, m/z 1076.7 [M2H]2,
positive ion, m/z 1078.7 [M1H]1; HRFABMS m/z
1078.7244 [M1H]1, calcd for C53H96N11O12 1078.7240
Similarly, N-methyl-d-leucine, N-methyl-d-isoleucine,
N-methyl-l-isoleucine, N-methyl-d-alloisoleucine, N-methyl-
l-alloisoleucine and N-methyl-b-alanine were prepared from
respective amino acids.
1
(D 10.4 mmu); H and 13C NMR (CDCl3) are listed in
1
l-Me-Val. H NMR (D2O) d 3.69 (1H, d, Jˆ4.5 Hz), 2.62
Table 2.
(3H, s), 2.20 (1H, dsept, Jˆ4.5, 7 Hz), 0.94 (3H, d, Jˆ7 Hz),
0.89 (3H, d, Jˆ7 Hz); 13C NMR (D2O) d 170.9 (s), 67.4 (d),
32.9 (q), 29.2 (d), 18.1 (q), 17.0 (q).
Theonellapeptolide IIe (7). A white solid (11.9 mg);
[a]3D0ˆ213.48 (cˆ0.6, CHCl3); IR (®lm) nmax 3326, 2962,
1751, 1633, 1538 cm21; LRFABMS, positive ion, m/z 1405
[M1H]1; HRFABMS m/z 1404.9473 [M1H]1, calcd for
C70H126N13O16 1404.9446 (D 12.7 mmu); 1H NMR
(500 MHz, CDCl3) d 7.90 (br), 7.70 (br), 7.53 (br), 7.16
(br), 6.52 (br), 6.14 (br), 3.96 (d, Jˆ15 Hz), 3.90 (d,
Jˆ15 Hz), 3.43 (s), 3.42 (s), 3.18 (s), 3.12 (s), 3.06 (s),
3.04 (s), 3.02 (s) 2.88 (s); 13C NMR (125 MHz, CDCl3) d
174.0, 173.9, 173.7, 173.1, 172.8, 172.5, 172.4, 170.6 (£2),
170.4, 170.2, 169.5, 169.3, 167.9, 71.7, 70.5, 64.5, 62.4,
59.9, 54.8, 53.6, 53.5, 52.1, 48.7, 47.7, 45.2, 42.6, 41.4,
37.4, 36.3, 36.0, 35.8, 34.6, 34.4, 33.2, 31.3, 33.1, 30.8,
29.5, 26.4, 24.9, 24.8, 24.6, 23.3, 23.2, 22.0, 21.8, 21.4,
20.0, 19.5, 18.7, 17.4, 17.2, 15.6, 15.2, 14.5, 14.3, 11.6,
11.4, 10.7, 9.8.
1
d-Me-Leu. H NMR (D2O) d 3.80 (1H), 2.60 (3H, s), 1.70
(1H, m), 1.65 (2H, m), 0.82 (6H).
l-allo-Me-Ile. 1H NMR (D2O) d 3.50 (1H, d, Jˆ4 Hz), 2.60
(3H, s), 1.90 (1H, m), 1.36 (1H, dq, Jˆ14, 7 Hz), 1.18 (1H,
dq, Jˆ14, 7 Hz), 0.88 (3H, d, Jˆ7 Hz), 0.82 (3H, t,
Jˆ7 Hz).
1
l-Me-Ile. H NMR (D2O) d 3.77 (1H, d, Jˆ3.5 Hz), 2.62
(3H, s), 1.92 (1H, m), 1.41 (1H, dq, Jˆ14, 7 Hz), 1.25 (1H,
dq, Jˆ14, 7 Hz), 0.86 (3H, d, Jˆ6.5 Hz), 0.81 (3H, t,
Jˆ7 Hz).
Complete acid hydrolysis of theonellapeptolides and
barangamides
Theonellapeptolide Ia (8). White crystalline solid; IR
(®lm) nmax 3326, 2962, 1733, 1681, 1633, 1538 cm21
;
LRFABMS, negative ion, 1389 [M2H]2. It was
identi®ed by 1H NMR comparison with an authentic
sample.
Each sample (0.02±0.5 mg) in 6N HCl (0.5 mL) was
heated in a glass tube sealed under N2 at 1108C for 24 h.
After the reaction mixture was cooled to ambient
temperature, it was concentrated to dryness. 1H NMR
spectrum of each hydrolysate was recorded. Amino acid
Theonellapeptolide Ie (9). A white solid (32.0 mg); IR
(®lm) nmax 3315, 2962, 1733, 1633, 1540 cm21
;
1
residues for each compound were identi®ed by H NMR
(D2O) comparison with that of each authentic sample as
shown below.
LRFABMS, positive ion, m/z 1419 [M1H]1. It was identi-
®ed by comparison of the NMR data with those of an
authentic sample.
1. b-Ala (£3), Me-Ala, allo-Ile, Me-Ile, allo-Me-Ile, Leu
(£2), Me-Leu, Thr, Val, Me-Val.
Preparation of N-methyl amino acids
2. b-Ala (£3), Ala, allo-Ile, Me-Ile, allo-Me-Ile, Leu (£2),
Except for commercially available d-Me-alanine, l-Me-
alanine, d/l-Me-valine, standard N-methyl amino acids
were prepared by Retro-Aza Diels±Alder reaction14 as
follows: To a solution of l-valine (100 mg, 0.85 mM) in
1.5 mL of H2O was added 70 mL (0.86 mM) of 37%
aqueous formaldehyde and 160 mL of cyclopentadiene
(1.95 mM) and the mixture was stirred vigorously under
N2 at room temperature for 16 h. After washing with
CH2Cl2, the solution was neutralized with 0.1 mL of 2 M
NaHCO3 and concentrated under reduced pressure. The
residue was extracted with CH2Cl2 (2 mL£3), dried over
MgSO4, and puri®ed by column chromatography on silica
gel eluting with CH2Cl2±MeOH (85:15) to furnish a dia-
stereomeric mixture of 2-azanorbornenes (43 mg), which
was used directly in the next reaction. To a solution of the
above adduct in 1.0 mL CHCl3 was added 1.0 mL of
tri¯uoroacetic acid and triethylsilane (160 mL, 0.99 mM).
The resulting homogeneous reaction mixture was stirred at
ambient temperature under N2 for 6 h. After removal of the
solvent by N2 stream, the yellow residue was dissolved in
CH2Cl2 (1.5 mL), treated with 1.0 mL of 10% HCl and
washed with CH2Cl2/EtOAc. The aqueous layer was ®ltered
and dried to furnish N-methyl-l-valine (23.5 mg).
Me-Leu, Thr, Val, Me-Val.
3. b-Ala (£3), Ala, allo-Ile, Me-Ile, allo-Me-Ile, Leu (£2),
Thr, Me-Val.
4. b-Ala (£3), Ala, allo-Ile, allo-Me-Ile, Leu (£2), Thr,
Me-Val (£2).
5. b-Ala (£3), Ala, Me-Ile, allo-Me-Ile, Leu (£2), Thr, Val,
Me-Val
6. b-Ala (£3), Ala, allo-Ile, Me-Ile, Leu (£2), Thr, Me-Val
(£2)
7. b-Ala (£2), Me-b-Ala, Ala, allo-Ile, Me-Ile, allo-Me-Ile,
Leu (£2), Me-Leu, Thr, Val, Me-Val
8. b-Ala (£3), Me-Ala, Me-Ile, allo-Me-Ile, Leu (£2),
Me-Leu, Thr, Val (£2), Me-Val
9. b-Ala (£2), Me-b-Ala, Me-Ala, allo-Ile, Me-Ile,
allo-Me-Ile, Leu (£2), Me-Leu, Thr, Val, Me-Val

M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
9091
Marfey analysis
Val), 18.03 (d-Me-Val), 22.30 (l-Me-Ile), 25.05 (d-allo-
Ile), 26.21 (d-Leu).
Each of the commercially available d- and l-amino acids
(0.3 mg) in 10 mL of water was treated with 50 mL of
FDAA (Marfey's reagent, Na-(2,4-dinitro-5-¯uorophenyl)-
l-alaninamide) solution in acetone (10 mg/mL) in the
presence of aqueous NaHCO3 solution (20 mL, 1 M), and
the mixture was heated at 408C for 1 h. After the reaction
mixture was cooled to room temperature, 10 mL of 2 M HCl
solution was added for neutralization and concentrated to
dryness. Acetone solution of the residue was ®ltered and
analyzed by HPLC. The aliquot (0.02±0.2 mg) of the each
acid hydrolysate described above was treated with FDAA
and analyzed by HPLC.
7. 8.15 (l-Thr), 9.27 (b-Ala), 9.55 (Me-b-Ala), 10.15
(l-Ala), 13.48 (l-Val), 17.18 (l-Me-Val), 22.23 (l-Me-
Ile), 25.01 (d-allo-Ile), 26.18 (d-Leu), 28.28 (d-allo-
Me-Ile).
8. 8.18 (l-Thr), 9.25 (b-Ala), 10.42 (l-Me-Ala), 13.42
(l-Val), 17.18 (l-Me-Val), 18.05 (d-Val), 22.31 (l-Me-
Ile), 26.21 (d-Leu), 28.25 (d-allo-Me-Ile).
9. 8.15 (l-Thr), 9.25 (b-Ala), 9.52 (Me-b-Ala), 10.12
(l-Me-Ala), 13.48 (l-Val), 17.25 (l-Me-Val), 22.29
(l-Me-Ile), 25.03 (d-allo-Ile), 26.15 (d-Leu), 28.22
(d-allo-Me-Ile).
HPLC analysis was carried out by using a reverse phase
column (COSMOSIL, 5C18-AR-II, 10£250 mm) at ambient
temperature, eluting with 60% mixed buffer in MeCN (¯ow
rate: 2 mL/min) and detecting by UV detector at 340 nm.
The mixed buffer was prepared by mixing aqueous solution
of sodium borate, potassium chloride, citric acid, and
tris(hydroxymethyl)aminomethane (each 25 mM) and
0.05 M HCl in 3:2 ratio (pH 2±3). Individual amino acid
peak was identi®ed by retention time as well as coinjection
with standard amino acid derivatives. The retention times of
the FDAA standards were 8.15 min for l-Thr, 9.10 min for
d-Thr, 8.15 min for l-allo-Thr, 8.40 min for d-allo-Thr,
9.25 min for b-Ala, 9.58 min for Me-b-Ala, 10.15 min for
l-Ala, 12.15 min for d-Ala, 10.42 min for l-Me-Ala,
12.40 min for d-Me-Ala, 13.45 min for l-Val, 18.00 min
for d-Val, 17.20 min for l-Me-Val, and 20.30 min for
d-Me-Val, 18.10 min for l-Ile, 25.25 min for d-Ile,
17.48 min for l-allo-Ile, 25.00 min for d-allo-Ile, 22.25
min for l-Me-Ile, 28.48 min for d-Me-Ile, 21.55 min for
l-allo-Me-Ile, 28.25 min for d-allo-Me-Ile, 18.45 min for
l-Leu, 26.20 min for d-Leu, 21.20 min for l-Me-Leu, and
29.00 min for d-Me-Leu.
d-Me-Leu was not detected in this analysis, but it was
1
con®rmed from the H NMR of complete acid hydrolysate
of individual peptolide. FDVA [Na-(2,4-dinitro-5-¯uoro-
phenyl)-d-valinamide] instead of FDAA was used as the
derivatization agent, which also enabled us to separate
l-Thr and l-allo-Thr with the above mentioned solvent
system.
Methanolysis of theonellapeptolides
To a solution of each sample (1±5 mg) in 1 mL of dry
MeOH was added 28% NaOMe (50 mL), and the solution
was stirred at room temperature for 6 h. The reaction
mixture was poured into ice water (1 mL) and extracted
with EtOAc (1 mL£3). The ethyl acetate extract was
washed with brine, dried over anhydrous MgSO4, and
concentrated. The product was puri®ed by ODS HPLC
with MeOH-MeCN-H2O (7.8:1:1.2).
1a. LRFABMS, negative ion, m/z 1435 [M2H]2, 1391
[M2C2H4O]2, positive ion, m/z 1437 [M1H]1, fragment
ions are shown in Fig. 4.
The observed amino acids and their retention times (min)
for FDAA derivatives of acid hydrolysate of 1±9 are as
follows:
2a. LRFABMS, negative ion, m/z 1421 [M2H]2, 1377
[M2C2H4O]2, positive ion, m/z 1445 [M1Na]1, 1423
[M1H]1, 1377, 1351, 1252, 1125, 1024, 1009, 953, 896,
840, 782, 712, 711, 637, 584, 528, 471, 413, 400, 342, 299,
172, 158.
1. 8.15 (l-Thr), 9.26 (b-Ala), 10.45 (l-Me-Ala), 13.40
(l-Val), 17.18 (l-Me-Val), 22.28 (l-Me-Ile), 25.05
(d-allo-Ile), 26.25 (d-Leu), 28.30 (d-allo-Me-Ile).
7a. LRFABMS, positive ion, m/z 1437 [M1H]1, fragment
ions are shown in Fig. 4.
2. 8.15 (l-Thr), 9.27 (b-Ala), 10.15 (l-Ala), 13.42 (l-Val),
17.15 (l-Me-Val), 22.25 (l-Me-Ile), 25.00 (d-allo-Ile),
26.26 (d-Leu), 28.32 (d-allo-Me-Ile).
8a. LRFABMS, negative ion, m/z 1421 [M2H]2, and 1377
[M2C2H4O]2, positive ion, m/z 1445 [M1Na]1, 1423
[M1H]1, 1351, 1264, 1252, 1151, 1125, 1080, 1024, 995,
953, 882, 840, 782, 712, 711, 641, 584, 542, 471, 429, 400,
340, 299.
3. 8.15 (l-Thr), 9.25 (b-Ala), 10.18 (l-Ala), 17.15 (l-Me-
Val), 22.20 (l-Me-Ile), 25.02 (d-allo-Ile), 26.22 (d-Leu),
28.28 (d-allo-Me-Ile).
4. 8.17 (l-Thr), 9.25 (b-Ala), 10.16 (l-Ala), 17.18 (l-Me-
Val), 25.01 (d-allo-Ile), 26.22 (d-Leu), 28.25 (d-allo-Me-
Ile).
9a. LRFABMS m/z 1451 [M1H]1, 1379, 1280, 1180, 1107,
1052, 1022, 967, 853, 796, 726, 725, 654, 598, 541, 484,
428, 344, 299, 172, 158.
5. 8.15 (l-Thr), 9.24 (b-Ala), 10.15 (l-Ala), 17.22 (l-Me-
Val), 18.10 (d-Val), 22.24 (l-Me-Ile), 26.20 (d-Leu), 28.28
(d-allo-Me-Ile).
Partial hydrolysis of theonellapeptolides
To a solution of each sample (1±5 mg) in 1 mL of dioxane
was added 50 mL of 2 M KOH solution in water, and the
6. 8.17 (l-Thr), 9.26 (b-Ala), 10.15 (l-Ala), 17.15 (l-Me-

9092
M. C. Roy et al. / Tetrahedron 56 (2000) 9079±9092
solution was stirred at room temperature for 3 h. The reac-
tion mixture was poured into ice water and acidi®ed with 1N
HCl, and extracted with EtOAc (1 mL£2). The combined
EtOAc extract was washed with brine, dried over MgSO4,
and concentrated. The crude product was puri®ed by ODS
PTLC with MeOH-H2O (9:1).
Acknowledgements
We thank Drs M. Kobayashi and S. Aoki (Osaka University)
for their collaboration in the specimen collection, donation
of authentic theonellapeptolides samples, and cytotoxicity
test; Drs Y. Ohshima and M. Satake (Tohoku University) for
recording HRFABMS; Dr R. Sakai (Kitasato University) for
recording some of LRFABMS; Dr M. Sakakibara, Dr Y.
Koezuka, Mr Y. Murakami (Kirin Brewery Co.) for the
immunosuppressive activity test and recording HRFABMS.
This work was partially supported by grant-in-aid (No.
09041184) from the Ministry of Education, Science, Sports,
and Culture of Japan.
1b. LRFABMS, positive ion, m/z 1423 [M1H]1, fragment
ions are shown in Fig. 4.
2b. LRFABMS, positive ion, m/z 1409 [M1H]1, 1237,
1110, 1009, 1008, 937, 895, 825, 711, 698, 628, 584, 514,
471, 400, 399, 330, 299, 257, 172, 144, 73.
7b. LRFABMS, positive ion, m/z 1423 [M1H]1, fragment
ions are shown in Fig. 4.
References
8b. LRFABMS, negative ion, m/z 1408 [M]2, 1389, 1153,
1110, 1079, 1061, 1009, 892, 792, 411 positive ion, m/z
1431 [M1Na]1, 1409 [M1H]1, 1391, 1166, 1079, 1061,
1009, 994, 976, 938, 882, 825, 785, 711, 697, 625, 584, 528,
467, 413, 400, 299, 328.
1. Doi, M.; Ishida, T.; Kobayashi, M.; Kitagawa, I. J. Org. Chem.
1991, 56, 3629±3632 and references cited therein.
2. Fusetani, N.; Matsunaga, S.; Matsumoto, H.; Takebayashi, Y.
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12620.
9b. LRFABMS, negative ion, m/z 1436 [M]2, 1390, 1194,
1035, 908, 837, 795 positive ion, m/z 1437 [M1H]1, 1196,
1107, 1037, 1023, 953, 910, 839, 797, 725, 711, 640, 526,
413, 400, 299, 328, 257, 172, 73.
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Methylation of 1, 2, 7, and 9.15 To a solution of each
sample (ca. 2 mg) in 300 mL of dry DMF was added
36 mg (155.2 mM) of Ag2O and 25 mL (405 mM) of MeI,
and the mixture was heated at 508C (oil bath) for 4 h. The
reaction mixture was diluted with DMF (500 mL) and
®ltered off. To the ®ltrate CH2Cl2 (2.5 mL) was added,
whereupon a white precipitate appeared, which was
removed by washing with KCN solution (5%, 2 mL).
Then the CH2Cl2 layer was washed with water several
times, dried over Na2SO4 and concentrated under N2 stream.
The solid product (10) was puri®ed by ODS HPLC
(MeOH±MeCN±H2O, 8:1:1 with 0.01% TFA).
6. Kobayashi, M.; Lee, N. K.; Shibuya, H.; Momose, T.;
Kitagawa, I. Chem. Pharm. Bull. 1991, 39, 1177±1184.
7. Kobayashi, M.; Kanzaki, K.; Katayama, S.; Ohashi, K.; Okada,
H.; Ikegami, S.; Kitagawa, I. Chem. Pharm. Bull. 1994, 42, 1410±
1415.
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W. T.; Pannell, L. K. J. Nat. Prod. 1998, 61, 724±728.
10. The sponge was identi®ed by Dr J. N. A. Hooper, Queensland
Museum, Brisbane, Australia. A voucher specimen (QMG 315201)
was deposited at the Queensland Museum, South Brisbane,
Queensland, Australia.
10. LRFABMS, positive ion, m/z 1517 [M1H]1. The same
amino acid residues, Me-b-Ala (£3), Me-Ala, Me-Ile, allo-
Me-Ile (£2), Me-Leu (£3), Me-Thr, Me-Val (£2), for 10
were identi®ed by 1H NMR (D2O) analysis of acid
hydrolysate of each sample of 10 prepared from 1, 2, 7,
and 9. The absolute con®guration of each amino acid
was con®rmed by Marfey analysis as l-Me-Ala, l-Me-Ile,
d-allo-Me-Ile (£2), and l-Me-Val (£2).
11. Roy, M. C.; Ohtani, I. I.; Tanaka, J.; Higa, T.; Satari, R.
Tetrahedron Lett. 1999, 40, 5373±5376.
12. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591±596.
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Â
15. Thomas, D. W.; Das, B. C.; Gero, S. D.; Lederer, E. Biochem.
Mixed lymphocyte reaction (MLR) assay
Biophys. Res. Commun. 1968, 32, 199±207.
The assay was carried out by Dr Y. Koezuka and his
coworkers, Kirin Brewery Co. Ltd.