Papuamides A-D, HIV-inhibitory and cytotoxic depsipeptides from the sponges Theonella mirabilis and Theonella swinhoei collected in Papua New Guinea

Article abstract of DOI:10.1021/ja990582o

The novel cyclic depsipeptides papuamides A (1), B (2), C (3), and D (4) have been isolated from Papua New Guinea collections of the sponges Theonella mirabilis and Theonella swinhoei. Their structures were determined by a combination of spectroscopic analysis and chemical degradation and derivatization studies. In addition to glycine, alanine, and threonine, these peptides contain a number of unusual amino acids including 3,4- dimethylglutamine, β-methoxytyrosine, 3-methoxyalanine, and 2,3- diaminobutanoic acid or 2-amino-2-butenoic acid residues. Papuamides A-D (1- 4) are also the first marine-derived peptides reported to contain 3- hydroxyleucine and homoproline residues. These peptides also contain a previously undescribed 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic acid moiety N-linked to a terminal glycine residue. Papuamides A (1) and B (2) inhibited the infection of human T-lymphoblastoid cells by HIV-1(RF) in vitro with an EC₅₀ of approximately 4 ng/mL. Compound 1 was also cytotoxic against a panel of human cancer cell lines with a mean IC₅₀ of 75 ng/mL.

Full text of DOI:10.1021/ja990582o

J. Am. Chem. Soc. 1999, 121, 5899-5909
5899
Papuamides A-D, HIV-Inhibitory and Cytotoxic Depsipeptides from
the Sponges Theonella mirabilis and Theonella swinhoei Collected in
Papua New Guinea
Paul W. Ford,† Kirk R. Gustafson,† Tawnya C. McKee,† Nobuharu Shigematsu,‡
Laura K. Maurizi,‡ Lewis K. Pannell,‡ David E. Williams,§ E. Dilip de Silva,§,#
Peter Lassota, Theresa M. Allen,| Rob Van Soest,∇ Raymond J. Andersen,*,§ and
Michael R. Boyd*,†
Contribution from the Laboratory of Drug DiscoVery Research and DeVelopment, DeVelopmental
Therapeutics Program, DiVision of Cancer Treatment and Diagnosis, Frederick Cancer Research and
DeVelopment Center, National Cancer Institute, Building 1052, Room 121,
Frederick, Maryland 21702-1201, and Departments of Chemistry and Earth & Ocean Sciences,
UniVersity of British Columbia, VancouVer, British Columbia, Canada V6T 1Z1
ReceiVed February 23, 1999
Abstract: The novel cyclic depsipeptides papuamides A (1), B (2), C (3), and D (4) have been isolated from
Papua New Guinea collections of the sponges Theonella mirabilis and Theonella swinhoei. Their structures
were determined by a combination of spectroscopic analysis and chemical degradation and derivatization studies.
In addition to glycine, alanine, and threonine, these peptides contain a number of unusual amino acids including
3,4-dimethylglutamine, â-methoxytyrosine, 3-methoxyalanine, and 2,3-diaminobutanoic acid or 2-amino-2-
butenoic acid residues. Papuamides A-D (1-4) are also the first marine-derived peptides reported to contain
3-hydroxyleucine and homoproline residues. These peptides also contain a previously undescribed 2,3-dihydroxy-
2,6,8-trimethyldeca-(4Z,6E)-dienoic acid moiety N-linked to a terminal glycine residue. Papuamides A (1)
and B (2) inhibited the infection of human T-lymphoblastoid cells by HIV-1RF in vitro with an EC50 of
approximately 4 ng/mL. Compound 1 was also cytotoxic against a panel of human cancer cell lines with a
mean IC50 of 75 ng/mL.
Sponges in the genus Theonella (order Lithistida) have been
a prolific source of structurally diverse, biologically active
peptides.1 Recent examples of Theonella peptides include
polytheonamides,2 cyclotheonamides,3 theonellapeptolides,4 the-
onellamides,5 theonegramides,6 keramamides,7 mozamides,8
mutoporins,9 microsclerodermins,10 cupolamide,11 oriamide,12
and cyclolithistide A.13 The biosynthetic origin of compounds
isolated from Theonella sponges has often been attributed to
symbiotic bacteria or microalgae. Recently, the peptide me-
tabolite theopalauamide14 was found localized in eubacteria that
live symbiotically within Theonella swinhoei. Many Theonella-
derived peptides demonstrate potent cytotoxicity,2,5,11,12 thrombin
inhibition,3 phosphatase inhibition,9 protease inhibition,3 or
antifungal properties.5,6,10,13,14 However, there have not been any
previous reports of peptides from Theonella that inhibit the
human immunodeficiency virus (HIV). The only sponge-
* To whom correspondence should be addressed. Michael R. Boyd:
phone, (301) 846-5391; fax, (301) 846-6919; e-mail, boyd@dtpax2.ncifcrf.gov.
Raymond J. Andersen: phone, (604) 822-4511; fax, (604) 822-4511; e-mail,
randersen@unixg.ubc.ca.
† National Cancer Institute.
‡ Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Diseases, Bethesda, MD 20892-0805.
§ University of British Columbia.
# Permanent address: Department of Chemistry, University of Colombo,
Colombo, Sri Lanka.
Oncology and Immunology Division, Building 200/4219A, Wyeth
Ayerst Research, 401 N. Middletown Road, Pearl River, New York, USA
10965.
(5) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Wa¨lchli, M. J. Am. Chem.
Soc. 1989, 111, 2582-2588. Matsunaga, S.; Fusetani, N. J. Org. Chem.
1995, 60, 1177-1181.
| Department of Pharmacology, University of Alberta, Edmonton, Alberta,
Canada T6G 2H7.
(6) Bewley, C. A.; Faulkner, D. J. J. Org. Chem. 1994, 59, 4849-4852.
(7) Kobayashi, J.; Itagaki, F.; Shigemori, H.; Takao, T., Shimonishi, Y
Tetrahedron 1995, 51, 2525-2532.
∇ Department of Coelenterates and Porifera, Institute for Systematics and
Population Biology (Zoologisch Museum), University of Amsterdam, P.O.
Box 94766, 1090 AT Amsterdam, The Netherlands.
(1) Faulkner, D. J. Nat. Prod. Rep. 1998, 15, 113-158. See also previous
reports in this series. Fusetani, N.; Matsunaga, S. Chem. ReV. 1993, 93,
1793-1806.
(8) Schmidt, E. W.; Harper, M. K.,; Faulkner, D. J. J. Nat. Prod. 1997,
60, 779-782.
(9) de Silva, E. D.; Williams, D. E.; Andersen, R. J.; Klix, H.; Holmes,
C. F. B.; Allen, T. M. Tetrahedron Lett. 1992, 33, 1561-1564.
(10) Schmidt, E. W.; Faulkner, D. J. Tetrahedron 1998, 54, 3043-3056.
(11) Bonnington, L. S.; Tanaka, J.; Higa, T.; Kimura, J.; Yoshimura,
Y.; Nakao, Y.; Yoshida, W. Y.; Scheuer, P. J. J. Org. Chem. 1997, 62,
7765-7767.
(2) Hamada, T.; Suyawara, T.; Matsunaga, S.; Fusetani, N. Tetrahedron
Lett. 1994, 35, 609-612.
(3) Fusetani, N.; Matsunaga, S.; Matsumoto, H.; Takebayashi Y. J. Am.
Chem. Soc. 1990, 112, 7053-7054. Lee, Y. A.; Hagihara, M.; Karmacharya,
R.; Albers, M. W,; Schreiber, S. L.; Clardy, J. J. Am. Chem. Soc. 1993,
115, 12619-12620. Nakao, Y.; Matsunaga, S.; Fusetani, N. Biorg. Med.
Chem. 1995, 3, 1115-1122. Nakao, Y.; Oku, N.; Matsunaga, S.; Fusetani,
N. J. Nat. Prod. 1998, 61, 667-670.
(12) Chill, L.; Kashman, Y.; Schleyer, M. Tetrahedron 1997, 53, 16147-
16152.
(13) Clark, D. P.; Carroll, J.; Naylor, S.; Crews, P. J. Org. Chem. 1998,
63, 8757-8764.
(14) Bewley, C. A.; Faulkner, D. J. Angew. Chem., Int. Ed. Engl. 1998,
37, 2162-2178. Schmidt, E. W.; Bewley, C. A.; Faulkner, D. J. J. Org.
Chem. 1998, 63, 1254-1258.
(4) Kobayashi, M.; Kanzaki, K.; Katayama, S.; Ohashi, K.; Okada, H.;
Ikegami, S.; Kitagawa, I. Chem. Pharm. Bull. 1994, 42, 1410-1415.
10.1021/ja990582o CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/15/1999

5900 J. Am. Chem. Soc., Vol. 121, No. 25, 1999
Ford et al.
derived, nonribosomal peptides previously reported to have anti-
HIV activity are the callipeltins,15 which were isolated from
the Lithistid sponge Callipelta sp. We report herein the isolation
and structure determination of papuamides A-D (1-4), HIV-
inhibitory and cytotoxic depsipeptides isolated from Papua New
Guinea collections of Theonella mirabilis and Theonella swin-
hoei.
solvent/solvent partitioning, reversed-phase flash chromatogra-
phy, gel permeation on Sephadex LH-20, and C18 reversed-
phase HPLC separations. The anti-HIV activity of T. mirabilis
was tracked to a mixture of peptides consisting primarily of
papuamides A (1) and B (2)18 while the cytotoxic activity of T.
swinhoei was traced to papuamides A-D (1-4).
Papuamide A (1), the most abundant peptide in both Theon-
ella samples, was isolated as an optically active amorphous
glass. A molecular formula of C66H105N13O21 was established
for 1 by high-resolution FABMS. Papuamide A (1) gave a sharp,
well-resolved peak when analyzed by reversed-phase HPLC
using a variety of solvent systems, and it gave a single clean
molecular ion in the HRFABMS. However, many of the
1
resonances in the 13C and H NMR spectra of 1 were doubled
or tripled. For example, in both DMSO-d6 and MeOH-d4, the
resonance ultimately attributed to H3 and H5 of the tyrosine
residue in 1 appeared as three doublets in the ratio 4:2:1. Other
NMR solvents, including pyridine-d5, DMF-d7, acetone-d6, and
a wide variety of combined solvent systems all provided spectra
with different peak ratios or broadened, poorly resolved
resonances that were not suitable for structural studies. Attempts
to simplify the NMR spectra by heating (50 °C) or adjusting
the pH by addition of TFA, pyridine, or Et3N did not alleviate
the problem. Eventually it was found that acceptable spectra
with only a single set of well-resolved resonances could be
obtained using solvent suppression and a mixture of CD3CN-
H2O (4:1) as the NMR solvent. Complete NMR spectral
assignments (Table 1) and the structural elucidation of 1 utilized
NMR data sets collected at three different concentrations, 3.0,
15.6 and 27.5 mM, in CD3CN-H2O (4:1). The doubling and
tripling of resonances observed in the NMR spectra of papua-
mide A (1) was attributed to the existence of slow conforma-
tional equilibria.
The peptide nature of 1 was apparent from a series of
exchangeable amide NH protons between δ 7.0 and δ 9.5 that
were coupled to either R-methine protons or R-methylene
protons between δ 3.7 and δ 5.3. In addition, 13 13C NMR
resonances observed between δ 181.0 and δ 169.0 had chemical
shifts appropriate for amide or ester carbonyls. A diacetate
derivative 5 was produced by sequential treatment of 1 with
Ac2O in MeOH followed by Ac2O in pyridine. Compound 5
also provided a single set of well-dispersed proton resonances
in the CD3CN-H2O (4:1) solvent mixture. Extensive NMR
analyses of compounds 1 and 5, including data from COSY,
TOCSY, ROESY, HSQC, HMBC, and HSQC-TOCSY experi-
ments, established the presence of alanine, threonine, and two
glycine residues. Seven uncommon amino acids were also
elucidated: homoproline (Hpr), â-methoxytyrosine (â-OMeTyr),
N-methylthreonine (NMeThr), 3-methoxyalanine (3-OMeAla),
3-hydroxyleucine (3-OHLeu), 3,4-dimethylglutamine (3,4-
DiMeGln), and 2,3-diaminobutanoic acid (Dab). In addition,
an amide-linked 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-
dienoic acid (Dhtda) moiety was elucidated. COSY and TOCSY
data helped establish the proton spin systems in the Dhtda
residue. The C4-C5 olefin was assigned a Z geometry on the
basis of coupling between H4 and H5 of approximately 11 Hz,
while ROESY correlations between H5 and H7 and between
the C6 Me group and both H3 and H8 established the C6-C7
double bond as E. The presence of a hydroxyl substituent at
C2 was revealed by its 13C chemical shift of δ 78.53, while
substitution at C2 with a methyl group was evident from HMBC
The extracts on which the present investigations were based
were from independent collections of T. mirabilis and T.
swinhoei made along the north coast of Papua New Guinea by
a contractor to the U.S. National Cancer Institute (NCI) and by
staff of the University of British Columbia (UBC), respectively.
In both cases, sponge samples were frozen immediately after
collection. Chemical investigations of T. mirabilis were initiated
at the NCI following observations that both the aqueous and
organic extracts exhibited activity in the NCI’s primary in vitro
anti-HIV screen.16 The UBC group, which has an ongoing
program to study cytotoxic metabolites from marine sponges,17
initiated its investigations based on observations that extracts
of T. swinhoei demonstrated potent in vitro cytotoxicity against
a panel of human cancer cell lines. Independent bioassay-guided
fraction efforts at NCI and UBC employed a combination of
(15) Zampella, A.; D’Auria, V. D.; Gomez Paloma, L.; Casapullo, A.;
Minale, L.; Debitus, C.; Henin, Y. J. Am. Chem. Soc. 1996, 118, 6202-
6209. D’Auria, M. V.; Zampella, A.; Gomez Paloma, L.; Minale, L.
Tetrahedron 1996, 52, 9589-9596.
(16) Boyd, M. R. In AIDS Etiology, Diagnosis, Treatment and PreVen-
tion; De Vita, V. T., Jr., Hellman, S., Rosenberg, S. A., Eds.; Lippincott:
Philadelphia, 1988; pp 305-319.
(17) For previous studies see: Williams, D. E.; Lassota, P.; Andersen,
R. J. J. Org. Chem. 1998, 53, 4838-4841. Coleman, J. E.; Dilip de Silva,
E.; Kong, F.; Andersen, R. J.; Allen, T. M. Tetrahedron 1995, 39, 10653-
10662. Kong, F.; Andersen, R. J.; Allen, T. M. J. Am. Chem. Soc. 1994,
116, 6007-6008.
(18) Subsequent mass spectrometric analysis of HPLC side fractions from
T. mirabilis revealed MH+ molecular ions at m/z 1400 and 1386 which are
consistent with the co-occurrence of papuamides C (3) and D (4),
respectively, as trace constituents of T. mirabilis.

Papuamides A-D
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5901
Table 1. 1H and 13C NMR Assignments for Papuamide A (1)a
δ
13C
1H
mult (J, Hz)
TOCSY
HMBCb
ROESY
Homoproline (Hpr)
CO
2
3
170.33
54.76
26.06
4.94
1.67
2.10
1.15
1.63
1.35
d (3.9)
m
m
m
m
1.15, 1.35, 1.63, 2.10
1.15, 1.35
1.15, 1.67
1.67, 1.69, 2.10
1.15, 1.35, 1.69, 2.10
1.15, 1.67, 1.69, 2.10
20.55, 26.06, 170.33
1.35, 1.63, 2.10, 4.07
1.15, 1.35
1.63, 4.94
1.35, 1.67
4
5
20.55
24.82
1.35, 2.10, 4.94
1.15, 1.67, 4.07, 4.24
4.94, 5.24
4.07
4.07
m
1.69
3.14
4.07
m
m
1.15, 2.10, 3.14, 4.07
1.67, 2.10, 4.07
1.67, 2.10, 3.14
6
44.24
54.76
br d (11.7)
1.35, 1.69, 3.14, 4.24
â-Methoxytyrosine (â-OMeTyr)
CO
R
173.22
52.07
5.24
4.24
3.03
7.20
6.76
t (9.3)
d (9.3)
s
4.24, 7.76
84.23, 168.75,
173.22
52.07, 56.81,
130.21, 173.22
84.23
1.35, 3.03, 4.07, 4.24
4.32, 7.20, 7.76
4.07, 5.24, 7.20, 7.76
â
84.38
5.24, 7.76
â-OMe
1
2, 6
56.81
128.88
130.21(2)
5.24, 7.20
d (8.3)
d (8.3)
6.76
7.20
84.23, 115.74,
130.21, 157.94
115.74, 128.88,
157.94
0.48, 3.03, 4.24, 5.24
6.76, 7.76
0.48, 7.20
3, 5
115.74(2)
157.94
4
NH
7.76
d (9.3)
4.24, 5.24
168.75
0.48, 3.87, 4.24, 4.32
5.24, 7.20
N-Methylthreonine (NMeThr)
1
2
3
4
168.75
64.11
63.66
19.64
4.32
3.87
0.48
m
d (6.4)
d (5.9)
0.48, 3.87
0.48, 4.32
3.87, 4.32
19.64, 63.66, 168.75
19.64, 30.91, 64.11
64.11
0.48, 3.87, 5.24, 7.76
0.48, 3.06, 4.32, 7.76
3.87, 4.32, 6.76, 7.20
7.76
N-Me
30.91
3.06
s
64.11, 174.41
3.87, 4.49
Alanine (Ala)
1
2
3
NH
174.41
47.73
15.39
4.49
1.36
7.12
m
1.36, 7.12
4.49, 7.12
1.36, 4.49
15.39, 174.41
47.73, 174.41
1.36, 3.06, 7.12
4.49, 7.12
d (10.7)
d (5.9)
15.39, 47.73, 171.04
1.36, 4.49, 8.27
Glycine 1 (Gly 1)
1
2
171.04
43.05
3.72
3.89
8.27
br d (15.6)
br d (15.6)
br s
3.89, 8.27
3.72, 8.27
3.72, 3.89
171.04
171.04
172.11
3.89, 8.27
3.72, 8.27
3.72, 3.83, 3.89, 4.18
7.12
NH
3-Methoxyalanine (3-OMeAla)
1
2
3
172.11
55.94
71.12
4.18
3.60
3.83
3.34
7.93
d (3.9)
br d (16.9)
br d (16.9)
s
3.60, 3.83, 7.93
3.83, 4.18, 7.93
3.60, 4.18, 7.93
71.12, 172.11
172.11
172.11
71.12
172.07
3.60, 3.83, 7.93, 8.27
3.34, 4.18
3.34, 4.18, 8.27
3.60, 3.83, 4.93
4.18, 4.26
3-OMe
NH
59.27
br s
3.60, 3.83, 4.18
3-Hydroxyleucine (3-OHLeu)
1
2
172.07
53.63
4.93
m
0.83, 5.23, 8.96
0.83, 0.98, 1.95, 3.34
5.23
0.84, 1.95, 4.93
0.83, 0.84, 4.93, 5.23
8.96
3
4
78.54
28.54
5.23
1.95
d (10.7)
m
0.84, 1.95, 4.93, 8.96
0.83, 0.84, 5.23
170.33
18.85, 78.54
5
5N
NH
18.44
18.85
0.84
0.83
8.96
d (6.4)
d (6.4)
br s
0.83, 1.95, 5.23
0.84, 1.95, 4.93
4.93, 5.23
18.85, 78.54
18.44, 78.54
1.95, 5.23
1.95, 4.93
1.95, 4.26
3,4-Dimethylglutamine (3,4-DiMeGln)
1
2
174.14
58.67
4.26
2.12
d (7.8)
m
0.98, 2.12, 8.69
37.52, 174.14
179.04
0.98, 2.12, 2.67, 7.93
8.96
0.98, 1.11, 2.67, 4.26
8.69
3
37.52
0.98, 4.26

5902 J. Am. Chem. Soc., Vol. 121, No. 25, 1999
Ford et al.
Table 1 (Continued)
δ
13C
1H
mult (J, Hz)
TOCSY
HMBCb
58.67
ROESY
3,4-Dimethylglutamine (3,4-DiMeGln)
2.12, 2.67, 4.26
3-Me
4
12.75
40.64
0.98
2.67
d (6.4)
m
2.12, 2.67, 4.26, 4.93
0.98, 1.11, 2.12, 4.26
7.10, 8.69
0.98, 1.11
12.75, 179.04
4-Me
5
2-NH
5-NH
15.08
179.04
1.11
d (6.8)
2.67
179.04
2.12, 2.67
8.69
6.58
7.10
br s
br s
br s
4.26, 4.65
7.10
6.58
171.60
2.12, 2.67
7.10
1.35, 2.67, 6.58
2,3-Diaminobutanoic acid (Dab)
1
2
171.60
55.27
4.65
br s
1.28, 8.69
48.77, 171.60
173.11
55.27
48.77, 55.27
173.11
3
4
48.77
15.27
3.96
1.28
8.69
m
br s
1.28
3.96, 4.65
2-NH
Threonine (Thr)
1
2
3
4
173.11
59.66
67.83
19.58
4.44
4.31
1.12
7.83
d (5.9)
m
d (9.3)
d (8.3)
1.12, 4.31, 7.83
1.12, 4.44, 7.83
4.31, 4.44, 7.83
1.12, 4.31, 4.44
173.11
19.58
1.12, 4.31, 7.83
1.12, 4.44
59.66, 67.83
172.20
4.31, 4.44, 7.83
1.12, 4.44, 8.31
NH
Glycine (Gly 2)
1
2
172.20
43.54
3.82
4.12
8.31
dd (16.6, 6.8)
dd (16.6, 6.3)
br s
4.12, 8.31
3.82, 8.31
3.82, 4.12
177.83
172.20, 177.83
177.83
4.12, 8.31
3.82, 8.31
3.82, 4.12, 7.83
NH
2,3-Dihydroxy-2,6,8-trimethyl-4,6-decadienoic Acid (Dhtda)
1
2
177.83
78.53
22.14
72.68
2-Me
3
1.20
4.80
s
72.68, 177.83
124.93, 138.51
4.80, 5.48
d (10.3)
5.48, 6.10
1.20, 1.75, 5.22, 5.48
6.10
1.20, 4.80, 6.10
1.75, 4.80, 5.22, 5.48
4
5
6
124.93
138.51
131.45
16.57
5.48
6.10
dd (10.3, 2.9)
dd (11.7, 2.9)
4.80, 6.10
4.80, 5.48
131.45
16.57, 72.68, 139.10
6-Me
1.75
5.22
2.32
br s
5.22
131.45, 138.51
139.10
16.57, 20.34, 30.54
34.67, 138.51
20.69, 131.45
2.32, 4.80, 6.10
7
8
139.10
34.67
d (9.5)
m
0.92, 1.21, 1.34, 1.75
2.32
0.82, 0.92, 1.21, 1.34
5.22
0.92, 1.21, 2.32, 4.80
6.10
0.82, 0.92, 1.21, 1.34
1.75, 5.22
8-Me
9
20.69
30.54
0.92
1.21
d (6.4)
m
1.21, 1.34, 2.32, 5.22
0.82, 0.92, 1.34, 2.32
30.54, 34.67, 139.10
34.67
1.34, 2.32, 5.22
0.82, 0.92, 2.32, 5.22
1.34
0.82
m
0.82, 0.92, 1.21, 2.32
5.22
1.21, 1.34, 2.32
139.10
0.82, 0.92, 2.32
1.21, 1.34, 2.32
10
12.00
t (6.4)
30.54, 34.67
a Spectra were recorded in CD3CN-H2O (4:1) at 500 MHz for 1H and 125 MHz for 13C and referenced to the residual solvent signal of CD3CN
1
(δH 1.93, δC 1.30). b Optimized for J ) 3.5 and J ) 8.5 Hz. Carbons correlated to proton resonances in the H column.
correlations observed between the methyl protons (δ 1.20) and
both C1 (δ 177.83) and C3 (δ 72.68). To the best of our
knowledge, the Dhtda group is an unprecedented structural
moiety. The only previous reports of the amino acid residues
â-OMeTyr and 2,3-DiMeGln have been as constituents of the
callipeltins.15 Papuamide A (1) also represents the first marine-
derived peptide to contain 3-OHLeu and Hpr residues.
The sequence of the amino acids, the location of the
decadienoic acid derivative, and the macrocyclic structure of
papuamide A (1) were established by detailed interpretation of
HMBC and ROESY experiments with compound 1 and its
diacetate derivative 5. HMBC correlations (Figure 1) observed
for papuamide A (1) defined most of the amino acid connec-
tivities; however, assignments of the 3-OHLeu and the C-
terminal Hpr residues were possible due to correlations that were
only observed with the diacetate 5. Inter-amino acid ROESY
correlations (Figure 2) helped confirm the amino acid sequence.
Again, several key confirmatory ROESY correlations were only
seen with compound 5. A HMBC correlation between the
N-terminal Gly-2 NH (δ 8.31) and the carbonyl (δ 177.83) of
the Dhtda moiety established that Dhtda was amide-linked to
Gly-2. The downfield chemical shift of the H3 carbinol proton
(δ 5.23) of 3-OHLeu suggested that the hydroxyl group was
part of a macrocyclic lactone. A HMBC correlation between
H3 and the Hpr carbonyl carbon (δ 170.33) confirmed cycliza-
tion via ester bond formation between the C-terminal Hpr
residue and the hydroxyl group of 3-OHLeu.
Several chemical degradations were undertaken to generate
small fragments of papuamide A (1) to help confirm the overall
structural assignment. First, papuamide A (1) was treated with
aqueous Et3N (5%) at 49 °C for 16 h. Removal of the reagents
in vacuo and fractionation of the reaction mixture via reversed-

Papuamides A-D
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5903
Figure 1. Key papuamide A (1) HMBC correlations. Dashed arrows
indicate correlations only observed in diacetate 5.
yield. Compound 7 was obtained as a clear oil that gave a [M
+ Na]+ ion at m/z 409.2324 in the HRFABMS appropriate for
a molecular formula of C19H34N2O6. Detailed analysis of the
1H, 13C, HMQC, and HMBC NMR data showed that 7 contained
a Gly residue, the 13-carbon Dhtda fragment previously
identified in papuamide A (1), and a 1,3-dihydroxy-2-aminobu-
tane (Dhab) moiety. Doubling of some resonances was also
apparent in the 1H NMR spectra of 7. HMBC correlations
observed from the Dhtha C2-Me singlet at δ 1.08 and the Gly
R-methylene proton resonances at δ 3.75-3.65 to a carbonyl
resonance at δ 175.4 confirmed that the Dhtda fragment was
attached via an amide bond to the Gly amino nitrogen. The Gly
NH (δ 7.29) and the Gly R-methylene (δ 3.75-3.65) resonances
also showed HMBC correlations to a carbonyl at δ 168.9,
confirming the existence of an amide bond between the amino
nitrogen of the Dhab moiety and the Gly carbonyl. ROESY
correlations confirmed the amide linkages identified via these
HMBC data. Isolation and identification of peptide fragments
6 and 7 helped confirm the overall amino acid sequence of
papuamide A (1).
The absolute stereochemistries of the amino acid constituents
of papuamide A (1) were determined following acid hydrolysis
(6 N HCl, 108 °C, 16 h) of the parent peptide. Commercially
available standards of alanine, threonine, 3-methoxyalanine, and
homoproline were available, while synthetic samples of 3-hy-
droxyleucine,19 2,3-diaminobutyric acid,20,21 and N-methyl-
threonine22 were analyzed. Stereochemical assignments were
made using a combination of GC-MS and LC-MS techniques
to compare the amino acids from papuamide A (1) with
appropriate standards. Treatment of the acid hydrolysate of 1
with Marfey’s reagent23 and LC-MS analysis of the resulting
1-fluoro-2,4-dinitrophenyl-5-L-alanine amide derivatives estab-
lished the presence of L-alanine, L-threonine, L-N-methylthreo-
nine, D-methoxyalanine, and L-homoproline. LC-MS analysis
of the amino acid derivatives generated by reaction of the
hydrolysate with 2,3,4,6-tetra-O-acetyl-â-D-glucopyranosyl isothio-
cyanate (GITC) confirmed these assignments. Treatment of the
hydrolysis mixture with acetyl chloride in 2-propanol followed
Figure 2. Key papuamide A (1) ROESY correlations. Dashed arrows
indicate correlations only observed in diacetate 5.
phase HPLC gave tripeptide 6 in moderate yield. Tripeptide 6
was obtained as an optically active clear oil that gave a [M +
H]+ ion in the HRFABMS at m/z 438.2235 appropriate for a
1
molecular formula of C21H32N3O7. Analysis of the H, 13C,
HMQC, and HMBC NMR data for 6 showed that it contained
Hpr, â-OMeTyr, and NMeThr residues. The doubling of
resonances observed in the NMR spectra of papuamide A (1)
was also apparent in the NMR spectra of the tripeptide 6. HMBC
correlations observed in compound 6 among the â-OMeTyr HR
resonance at δ 5.19, the â-OMeTyr NH resonance at δ 9.17,
the NMeThr H2 resonance at δ 3.27, and the NMeThr carbonyl
resonance at δ 164.5 demonstrated an amide bond between the
â-OMeTyr amino nitrogen and the carbonyl of the NMeThr
residue. In addition, the â-OMeTyr HR proton resonance at δ
5.19, the â-OMeTyr Hâ proton resonance at δ 4.31, and the
Hpr H2 proton resonance at δ 5.14 all showed HMBC
correlations to an amide carbonyl resonance at δ 169.6,
demonstrating the presence of an amide bond between the Hpr
tertiary amino nitrogen and the â-OMeTyr carbonyl. The Hpr
H2 proton resonance at δ 5.14 showed an HMBC correlation
to a carbonyl resonance at δ 172.0 that was assigned to the
free terminal carboxylic acid. ROESY and NOE data confirmed
the connectivities assigned by the HMBC data for tripeptide 6.
The second chemical degradation reaction involved treating
papuamide A (1) with excess NaBH4 in aqueous 2-propanol at
room temperature. Purification of the crude reaction product
via reversed-phase HPLC gave the previously identified tri-
peptide 6 and the new degradation product 7, both in very low
(19) Nagamitsu, T.; Sunazuka, T.; Tanaka, H.; Omura, S.; Sprengeler,
P. A.; Smith, A. B., III. J. Am. Chem. Soc. 1996, 118, 3584-3590.
(20) Schmidt, U.; Mundinger, K.; Riedl, B.; Haas, G.; Lau, R. Synthesis
1992, 1201-1202.
(21) (2S,3S)-Diaminobutyric acid for comparative purposes was also
obtained from the acid hydrolysate of aciculitin B. Bewley, C. A.; He, H.;
Williams, D. H.; Faulkner, D. J. J. Am. Chem. Soc. 1996, 118, 4314-
4321.
(22) Beulshausen, T.; Groth, U.; Scho¨llkopf, U. Liebigs Ann. Chem. 1992,
523-526.
(23) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.

5904 J. Am. Chem. Soc., Vol. 121, No. 25, 1999
Ford et al.
by trifluoroacetic anhydride in CH2Cl2 gave a mixture of
trifluoroacetyl isopropyl ester derivatives which were analyzed
by GC-MS using a Chirasil-L-Val capillary column. Compari-
son with appropriate standards confirmed the presence of
(2R,3R)-3-hydroxyleucine and (2S,3R)-diaminobutanoic acid.
Comparison by GC-MS of the trifluoroacetyl isopropyl esters
and LC-MS of both the FDAA and GITC derivatives of 3,4-
dimethylglutamic acid in the acid hydrolysate of papuamide A
(1) with those of the same derivatives from the hydrolysate of
authentic callipeltin A15 showed that the 3,4-dimethylglutamic
acid derived from these two peptides was indistinguishable. In
addition, GC-MS and LC-MS analysis of the 3,4-dimeth-
ylpyroglutamic acid, which also forms under these hydrolysis
conditions,15 indicated that the 3,4-DiMeGln residue in papua-
mide A (1) is identical to that in callipeltin A. This allowed
assignment of its configuration as (3S,4R)-dimethyl-L-glutamine.
It was not possible to isolate or characterize the â-methoxy-
tyrosine and 2,3-dihydroxy-2,6,8-trimethyldeca-(4E,6Z)-dienoic
acid moieties in the acid hydrolysate of 1, and thus their
stereochemistries were not assigned. Attempts to make ap-
propriate derivatives of compound 1, compound 2, and the
diacetate 5 were also unsuccessful due to decomposition of the
starting materials.
of the Dab residue in 1 and 2 were not apparent in the spectra
of 3 and 4. Instead there were resonances appropriate for a
2-amino-2-butenoic acid (Aba) residue. Thus, the Aba olefinic
methyl resonance at δ 1.66 (H4) was correlated in the COSY
spectrum of 4 to an olefinic proton at δ 6.67 (H3). This olefinic
proton resonance was in turn correlated in the HMQC spectrum
to a carbon resonance at δ 135.4 (C3), and the Aba olefinic
methyl resonance at δ 1.66 was correlated to C2 (δ 129.3) and
C3 (δ 135.4) in the HMBC spectrum. Additional HMBC
correlations were observed between the Aba carbonyl resonance
at δ 166.7 (C1) and both the 3,4-DiMeGln NH resonance at δ
8.00 and the Aba H3 resonance at δ 6.67. These latter HMBC
correlations confirmed the amide linkage between the amino
nitrogen of the 3,4-DiMeGln residue and the Aba carbonyl. The
Aba NH resonance of papuamide D (4) at δ 9.10 and the Thr
H2 resonance at δ 4.44 both showed HMBC correlations to a
carbonyl resonance in the region δ 172.5-172.7. This set of
HMBC correlations was consistent with an amide bond between
the Aba R-amino nitrogen and the Thr carbonyl. On the basis
of the NMR and MS evidence, papuamides C and D were
assigned the structures 3 and 4, respectively. Confirmation of
the proposed structures for 3 and 4 came from conversion of
papuamide A (1) to papuamide C (3) via Hofmann elimination
using excess MeI in the presence of K2CO3. Unfortunately,
ROESY, NOESY, or difference NOE data failed to provide
unambiguous proof for the configuration of the Aba olefins in
papuamide C (3) or D (4). Acid hydrolysis and amino acid
analysis by a combination of GC-MS and LC-MS techniques,
as described above, revealed that the absolute stereochemistries
of the amino acid constituents of papuamides C (3) and D (4)
were identical to those found in papuamides A (1) and B (4).
Papuamide A (1) and B (2) were evaluated in a tetrazolium-
based assay24 that measures the ability of test compounds to
The molecular formula of papuamide B (2) was established
as C65H103N13O21 by HRFABMS and confirmed by extensive
NMR analyses. It only differed from the molecular formula of
1
papuamide A (1) by the loss of CH2. The H and 13C NMR
spectral data of 2 were also very similar to those recorded for
1. However, it was possible to obtain well-resolved spectra of
2 in a variety of NMR solvents including CD3OH. Detailed
interpretation of 1-D and 2-D NMR data sets allowed the
complete spectral assignment (Table 2) for papuamide B (2).
While the presence of two OMe groups was evident, appropriate
NMR resonances for an N-methyl group were lacking in 2. This
suggested that the NMeThr in papuamide A (1) was simply
replaced with a Thr residue in papuamide B (2). Amino acid
analysis by GC-MS of the acid hydrolysate of 2 confirmed
the presence of 2 equiv of L-threonine. The structure of
compound 2 was ultimately elucidated through a similar series
of NMR experiments and by LC-MS and GC-MS character-
ization of its constituent amino acids, as described above for
papuamide A (1). Aside from substitution of Thr for the
NMeThr found in 1, the amino acid composition, sequence,
stereochemistries, point of lactonization, and attachment of the
Dhtda group in papuamide (2) were identical to those in
papuamide A (1).
protect CEM-SS T-cell cultures from infection by HIV-1RF
.
Increasing concentrations of compound 1 or 2 were added to
cell cultures coincident with the addition of HIV. At day six
post-infection, cultures treated with papuamide A (1) exhibited
a concentration-dependent increase in cellular viability, indicat-
ing an inhibition of productive infection relative to control
cultures, with an EC50 (50% effective concentration) of 3.6 ng/
mL. A cytotoxic IC50 (concentration inhibiting growth or
viability of uninfected control cells by 50%) was measured for
1 at 74 ng/mL. The HIV-inhibitory and cytotoxic activities of
papuamide B (2) in this assay were virtually identical to those
observed for papuamide A (1). Papuamide A (1) also produced
a characteristic cytotoxicity profile when tested against a panel
of human cancer cell lines, revealing a relative sensitivity of
multi-drug-resistant cell lines and a relative resistance of
leukemia cell lines. The mean IC50 of papuamide A (1) against
the human cancer cell line panel was 75 ng/mL.
Papuamides C (3) and D (4) were obtained as optically active
colorless glasses whose HRFABMS data were consistent with
molecular formulas of C66H103N12O21 and C65H101N12O21,
1
respectively. Comparison of the H, 13C, HMQC, and HMBC
Experimental Section
NMR data obtained for 3 and 4 with those of 1 and 2 indicated
that the two compounds were closely related to papuamides A
(1) and B (2). The relationship between papuamides C (3) and
D (4) was the same as that of 1 and 2 in that papuamide C (3)
was N-methylated at the amino nitrogen of threonine, while
papuamide D (4) was not.
The1H NMR spectra of papuamides C (3) and D (4) were
very poorly resolved; however, the HMBC and COSY data
collected for papuamide D (4) proved to be useful in elucidating
the gross structures. Preliminary analysis of the NMR data
revealed that the structures of papuamides C (3) and D (4)
differed from those of 1 and 2 at the Dab residue. NMR
resonances for the C2/H2 R-methine and the C3/H3 â-methine
General Procedures. Optical rotations were measured on a Perkin-
Elmer 241 polarimeter. UV spectra were recorded on a Beckman DU
640 spectrophotometer. IR spectra were obtained from neat samples
on KCl disks in a Perkin-Elmer Spectrum 2000 FT-IR spectrometer.
NMR spectra were recorded on a Varian Unity INOVA-500 NMR
spectrometer. NMR spectra were referenced to residual signals from
the NMR solvent. High-resolution fast atom bombardment (HRFAB),
electron impact, and chemical ionization mass spectra were acquired
on a JEOL SX102 mass spectrometer operated at an accelerating voltage
of 10 kV and 1000 resolution. Mass spectral data were acquired by a
JEOL XMS data system. Electrospray mass spectra were acquired on
a Hewlett-Packard HP1100 integrated LC-MS system.
(24) Gulakowski, R. J.; McMahon, J. B.; Staley, P. G.; Moran, R. A.;
Boyd, M. R. J. Virol. Methods 1991, 33, 87-100.

Papuamides A-D
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5905
Table 2. 1H and 13C NMR Assignments for Papuamide B (2)a
δ
13C
1H
mult (J, Hz)
TOCSY
HMBCb
ROESY
Homoproline (Hpr)
CO
2
171.33
53.33
5.26
d (7.8)
1.56, 1.70, 1.72, 2.10
3.30, 4.02
21.35, 27.45, 171.33
1.72, 2.10
3
27.45
1.70
2.10
m
m
2.10, 5.26
1.56, 1.70, 3.30, 4.02
5.26
1.72, 2.10, 4.02, 5.26
1.56, 1.70, 3.30, 4.02
5.26
1.50, 1.70, 1.72, 5.26
1.50, 2.10, 4.02, 5.26
4
21.35
1.56
1.72
m
m
5
6
26.24
44.47
1.50
1.70
3.30
m
m
m
3.30
1.72, 2.10, 3.30, 4.02
2.10, 3.30
1.50, 1.70, 4.02
1.72, 2.10, 3.30, 4.02
1.50, 1.70, 1.72, 2.10
4.02, 5.26
53.33
4.02
m
1.70, 1.72, 2.10, 3.30
5.26
1.50, 1.72, 3.30, 4.37
â-Methoxytyrosine (â-OMeTyr)
CO
R
171.07
53.50
5.18
4.37
3.10
7.18
6.75
t (9.4)
d (9.8)
s
4.37, 7.98
84.74
3.10, 4.02, 4.37, 7.18
7.98
â
84.74
5.18, 7.98
53.50, 56.91, 130.83
3.10, 4.02, 5.18, 7.18
7.98
â-OMe
1
2, 6
56.91
129.31
130.83(2)
4.37, 5.18, 7.18
d (8.3)
d (8.3)
6.75
7.18
84.74, 116.25
130.83, 158.91
116.25, 129.31
158.91
0.56, 3.10, 4.37, 5.18
6.75
7.18
3, 5
116.25(2)
158.91
4
NH
7.98
d (9.3)
m
4.37, 5.18
3.98, 4.37, 5.18
N-Methylthreonine (NMeThr)
1
2
170.98
60.18
3.98
0.56, 3.78, 7.81
67.92, 170.98
174.42
0.56, 7.72, 7.81, 7.98
3
4
NH
67.92
19.54
7.81
3.78
0.56
m
d (6.4)
d (9.3)
0.56, 3.98, 7.81
3.78, 3.98
3.78, 3.98
0.56, 7.72, 7.81
3.78, 3.98, 7.18
1.42, 3.78, 3.98, 4.29
60.18, 67.92
Alanine (Ala)
1
2
3
NH
174.42
50.75
18.00
4.29
1.42
7.72
q (6.8)
d (7.3)
d (7.3)
1.42, 7.72
4.29, 7.72
1.42, 4.29
18.00, 174.42
50.75, 174.42
1.42, 7.72, 7.81
4.29, 7.72, 7.81
1.42, 3.78, 3.98, 4.29
Glycine 1 (Gly 1)
1
2
172.06
43.85
3.70
4.02
8.51
m
m
t (5.9)
4.02, 8.51
3.70, 8.51
3.70, 4.02
172.06
172.06
4.02, 8.51
3.38, 3.70, 8.51
3.70, 4.02, 4.42
7.12
NH
3-Methoxyalanine (3-OMeAla)
1
2
3
172.44
55.41
71.88
4.42
3.75
3.81
3.38
8.21
m
m
m
s
3.75, 3.81, 8.21
3.81, 4.42, 8.21
3.75, 4.42, 8.21
172.44
3.75, 3.81, 8.21, 8.50
4.42, 8.21
4.42, 8.21
4.02
3-OMe
NH
59.31
71.88
d (6.8)
3.75, 3.81, 4.42
172.44
0.92, 3.75, 3.81, 4.42
4.98, 5.26, 8.78
3-Hydroxyleucine (3-OHLeu)
1
2
3
4
5
5N
NH
172.74
55.75
79.41
30.06
18.97
19.39
4.98
5.26
1.98
0.92
0.93
8.78
d (4.4)
d (7.8)
m
d (6.4)
d (6.4)
d (4.4)
0.93, 1.98, 5.26, 8.78
4.98, 8.78
0.92, 0.93, 4.98
1.98
1.98, 4.98
4.98, 5.26
172.74
0.93, 1.98, 5.26, 8.21
0.92, 0.93, 4.98, 8.21
0.92, 0.93, 4.98, 8.78
1.98, 5.26, 8.21, 8.78
1.98, 4.98, 5.26, 8.78
0.92, 0.93, 1.10, 1.98
4.20, 8.21
18.97, 19.39, 171.33
18.97, 19.39, 79.41
79.41
79.41
3,4-Dimethylglutamine (3,4-DiMeGln)
1
2
3
174.13
58.68
37.50
4.20
2.28
d (9.8)
m
1.10, 2.28, 9.54
1.10, 2.66, 4.20, 9.54
37.50
14.66, 118.14
2.28, 8.78
1.10, 1.22, 2.66, 4.20
9.54

5906 J. Am. Chem. Soc., Vol. 121, No. 25, 1999
Ford et al.
Table 2 (Continued)
δ
13C
1H
mult (J, Hz)
TOCSY
HMBCb
ROESY
3,4-Dimethylglutamine (3,4-DiMeGln)
3-Me
4
4-Me
5
15.98
43.93
14.66
1.10
2.66
1.22
d (6.8)
dq (7.3, 2.9)
d (7.3)
2.28, 4.20
1.22, 2.28
2.66
37.50, 43.93, 58.68
15.98, 37.50, 181.14
37.50, 43.93, 181.14
2.28, 2.66, 7.60, 8.78
1.10, 1.22, 2.28, 7.60, 9.54
2.28, 2.66
181.14
2-NH
5-NH
9.54
7.00
7.60
br s
br s
br s
2.28, 4.20
2.28, 2.66, 3.80, 4.52
7.60
1.10, 2.66, 7.00
43.93
2,3-Diaminobutanoic Acid (Dab)
1
2
3
4
171.87c
55.72
49.15
15.80
4.52
3.80
1.31
8.51
d (7.3)
m
d (6.8)
d (8.3)
1.31, 3.80, 8.51
1.31, 4.52, 8.51
3.80, 4.52
9.54
8.51, 9.54
3.80
55.72
49.15, 55.72
2-NH
3.80, 4.52
3.80, 4.45
Threonine (Thr)
1
2
3
4
174.22c
60.02
68.39
20.06
4.45
4.24
1.19
7.91
m
1.19, 4.24, 7.91
1.19, 4.45, 7.91
4.24, 4.45
1.19, 4.24, 7.91, 8.51
1.19, 4.45, 7.91
4.24, 4.45, 7.91
dd (6.4, 3.4)
d (6.4)
d (7.8)
60.02, 68.39
172.16
NH
4.24, 4.45
1.19, 4.06, 4.24, 4.45
Glycine (Gly 2)
1
2
172.16
43.84
3.86
4.06
8.50
d (17.1)
d (17.1)
t (5.9)
4.06, 8.50
3.86, 8.50
3.86, 4.02
172.16, 179.33
172.16, 179.33
179.33
4.06
3.86, 7.91, 8.50
1.26, 4.06
NH
2,3-Dihydroxy-2,6,8-trimethyl-4,6-decadienoic Acid (Dhtda)
1
2
179.33
78.72
2-Me
3
4
5
22.74
73.08
125.93
139.07
132.17
17.00
1.26
4.79
5.49
6.16
s
73.08, 78.72, 179.33
125.93, 139.07
132.17
5.49, 8.51
5.49
1.26, 1.80, 4.79, 6.16
1.80, 5.26, 5.49
d (10.3)
t (10.3)
5.49, 6.16
4.79, 6.16
4.79, 5.49
17.00, 73.08, 139.67
6
6-Me
1.80
5.26
2.36
0.97
1.28
1.39
0.86
br s
5.26
132.17, 139.07
139.67
17.00, 31.45, 35.63
139.07
2.36, 5.26, 5.49
1.80, 6.16
7
139.67
35.63
21.07
31.45
d (7.8)
m
0.86, 0.97, 1.28, 1.39
1.80, 2.36
0.86, 0.97, 1.28, 1.39
5.26
0.86, 1.28, 1.39, 2.36
5.26
0.86, 0.97, 2.36, 5.26
8
21.07, 139.67
0.97, 1.28, 1.39, 1.80
1.39, 2.36
8-Me
9
d (6.8)
m
31.45, 35.63, 139.67
12.49, 21.07, 35.63
139.67
12.49, 21.07, 35.63
139.67
0.86, 2.36
dd (11.3, 7.3)
0.86, 0.97, 2.36, 5.26
0.86, 0.97, 2.36
10
12.49
t (7.8)
0.97, 1.28, 1.39, 2.36
31.45, 35.63
1.28, 1.39
1
a Spectra were recorded in CD3OH at 500 MHz for H and 125 MHz for 13C and referenced to the residual solvent signal (δH 3.30, δC 49.00).
1
b Optimized for J ) 3.5 and J ) 8.5 Hz. Carbons correlated to proton resonances in the H column. c Assignments are interchangeable.
Collection, Extraction, and Purification. Specimens of T. mira-
bilis25 were collected in 1993 from a depth of -10 m near Madang
Harbor on the north coast of Papua New Guinea and frozen immediately
after collection. Frozen sponge (1359 g wet wt) was mixed with dry
ice and ground to a fine powder. Extraction with H2O and lyophilization
of the resulting solution provided 57 g of aqueous extract. The sponge
material was then freeze-dried and extracted with MeOH-CH2Cl2 (1:
1) and 100% MeOH to give 2.5 g of organic extract. A 32 g portion of
the aqueous extract was separated by vacuum liquid chromatography
on wide-pore (275 Å) C4 packing eluted with an aqueous-MeOH step
gradient. The material that eluted with H2O-MeOH (2:1) (308 mg)
was fractionated on Sephadex LH-20 with MeOH-H2O (7:3) to give
93 mg of a peptide rich sample. Final purification was achieved by
reversed-phase C18 HPLC using a linear gradient with increasing
concentrations of CH3CN in H2O (0.05% trifluoroacetic acid, vol/vol)
to give 12 mg of papuamide A (1) and 5 mg of papuamide B (2). A
portion of the organic extract (1.5 g) was separated by a modified
Kupchan solvent-solvent partition protocol.26 The aqueous-soluble
fraction from this protocol (793 mg) was applied to a C18 vacuum flash
chromatography column and eluted with increasing concentrations of
MeOH in H2O. Fractions eluted with MeOH-H2O (1:1) and 100%
MeOH were combined and purified by Sephadex LH-20 and C18 HPLC,
in a manner similar to that described above for the aqueous extract, to
give an additional 5 mg of 1 and 3 mg of 2.
Specimens of T. swinhoei (Gray, 1867)25 were collected at a depth
of -10 m from vertical walls northeast of Kranket Island, Madang
Lagoon, Papua New Guinea, in January 1994. Freshly collected sponge
was frozen on site and transported to Vancouver over dry ice. A voucher
sample of the sponge has been deposited in the Zoological Museum at
the University of Amsterdam. Approximately 1 kg of lyophilized sponge
was cut into small pieces and immersed in and subsequently extracted
repeatedly with MeOH (3 × 300 mL). The combined MeOH extracts
(26) Grode, S. H.; James, T. R.; Cardellina, J. H., II; Onan, K. D. J.
Org. Chem. 1983, 48, 5203-5207.
(27) Bokesch, H. R.; Groweiss, A.; McKee, T. C.; Boyd, M. R. J. Nat.
Prod. in press.
(25) Taxonomic identifications were made by R. Van Soest.

Papuamides A-D
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5907
were concentrated in vacuo and then partitioned between H2O (500
mL) and EtOAc (3 × 150 mL). The H2O extract was evaporated to
dryness in vacuo to yield 23.45 g of a red-orange amorphous solid as
fraction A. The dried EtOAc-soluble portion of the original crude
MeOH extract was taken up in 4:1 MeOH-H2O (466 mL) and extracted
repeatedly with CH2Cl2 (4 × 60 mL). The combined CH2Cl2 extracts
were concentrated to yield a red oil designated fraction C, and the
MeOH-H2O-soluble portion was evaporated to give fraction B.
Many of the carbons appeared as multiple resonances. Assignments
based on HMQC, HMBC, COSY, and ROESY data.
Papuamide D (4). Clear glass; [R]25 ) +16.1° (c 0.68, MeOH);
D
UV [4:1 CH3CN-H2O] λmax 226 (ꢀ 18 490), 274 (ꢀ 2272) nm;
HRFABMS provided m/z 1385.7157 (M + H)+, calculated 1385.7204
for C65H101N12O21, and m/z 1407.7029 (M + Na)+, calculated 1407.7024
1
for C65H100N12O21Na; H NMR (500 MHz, 4:1 CD3CN-H2O) δ Hpr
H-2, 5.07 (1H); H-3, 1.57 (1H) and 2.11 (1H); H-4, 1.63 (2H); H-5,
1.27 (1H) and 1.66 (1H); H-6, 3.10 (1H) and 4.05 (1H); â-OMeTyr
H-R, 5.18 (1H); H-â, 4.26 (1H); OMe on C-â, 3.02 (3H); H-2 and
H-6, 7.16 (2H); H-3 and H-5, 6.74 (2H); NH, 7.76 (1H); Thr 1 H-2,
3.81 (1H), H-3, 3.66 (1H); H-4, 0.45 (3H); NH, 7.86; Ala H-2, 4.17
(1H); H-3, 1.38 (3H); NH, 7.14; Gly 1 H-2, 3.48 (1H) and 4.08 (1H);
NH, 8.23 (1H); 3-OMeAla H-2, 4.17 (1H); H-3, 3.61 (1H) and 3.73
(1H); OMe on C-3, 3.28 (3H); NH, 7.96 (1H); 3-OHLeu H-2, 4.99
(1H); H-3, 5.23 (1H); H-4, 1.90 (1H); H-5, 0.82 (3H); H-6, 0.77 (3H);
NH, 8.57 (1H); 3,4-DiMeGln H-2, 4.25 (1H); H-3, 2.03 (1H); Me on
C-3, 0.92 (3H); H-4, 2.50 (1H); Me on C-4, 1.05 (3H); NH on C-2,
8.00 (1H); Aba H-3, 6.67 (1H); H-4, 1.66 (3H); NH, 9.10 (1H); Thr
2 H-2, 4.44 (1H); H-3, 4.27 (1H); H-4, 1.15 (3H); NH, 7.83 (1H); Gly
2 H-2, 3.79 (1H) and 4.05 (1H); NH, 8.32 (1H); Dhtda Me on C-2,
1.18 (3H); H-3, 4.77 (1H); H-4, 5.45 (1H); H-5, 6.10 (1H); Me on
C-6, 1.72 (3H); H-7, 5.18 (1H); H-8, 2.30 (1H); Me on C-8, 0.88 (3H);
H-9, 1.15 (1H) and 1.31 (1H); H-10, 0.77 (3H); 13C NMR (125 MHz,
4:1 CD3CN-H2O) δ Hpr CO, 170.7; C-2, 53.9; C-3, 26.7; C-4, 21.4;
C-5, 25.5; C-6, 44.7; â-OMeTyr CO, 171.9; C-R, 52.9; C-â, 84.3;
OMe on C-â, 57.3; C-1, 129.3; C-2 and C-6, 130.4; C-3 and C-5, 116.3;
C-4, 157.9; Thr 1 C-1, 170.7; C-2, 60.8; C-3, 66.9; C-4, 19.2; Ala
C-1, 174.9; C-2, 50.3; C-3, 17.4; Gly 1 C-1, 172.0; C-2, 42.9;
3-OMeAla C-1, 172.6; C-2, 57.1; C-3, 71.1; OMe on C-3, 59.3;
3-OHLeu C-1, 172.7; C-2, 55.6; C-3, 79.8; C-4, 28.9; C-5, 19.2; C-6,
18.9; 3,4-DiMeGln C-1, 175.0; C-2, 58.7; C-3, 37.8; Me on C-3, 13.1;
C-4, 41.1; Me on C-4, 15.5; C-5, 179.8; Aba C-1, 166.7; C-2, 129.3;
C-3, 135.4; C-4, 13.6; Thr 2 C-1, 172.7; C-3, 59.9; C-3, 69.0; C-4,
19.8; Gly 2 C-1, 172.5; C-2, 43.7; Dhtda C-1, 178.6; C-2, 78.8; Me
on C-2, 22.4; C-3, 73.0; C-4, 124.8; C-5, 139.2; C-6, 131.8; Me on
C-6, 16.9; C-7, 139.8; C-8, 35.0; Me on C-8, 20.9; C-9, 30.9; C-10,
12.4. Many of the carbons appeared as multiple resonances. Assign-
ments based on HMQC, HMBC, COSY, and ROESY data.
Fractionation of the aqueous extract, fraction A, by sequential
application of (i) C18 reversed-phase flash chromatography (gradient
elution: H2O to MeOH), (ii) Sephadex LH-20 (eluent: MeOH), (iii)
C18 reversed-phase HPLC (eluent: 2:3 CH3CN-aqueous 0.05% TFA),
and (iv) C18 reversed-phase HPLC (eluent: 42.5:57.5 CH3CN-aqueous
0.05% TFA) gave pure papuamide A (1) (57 mg) and papuamide B
(2) (6.4 mg) as clear glasses. Separation of the CH2Cl2 extract, fraction
C, via sequential application of (i) C18 reversed-phase flash chroma-
tography (gradient elution: 1:1 MeOH-H2O to MeOH), (ii) Sephadex
LH-20 chromatography (eluent: MeOH), and (iii) C18 reversed-phase
HPLC (eluent: 42.5:57.5 CH3CN-aqueous 0.05% TFA) gave pure
papuamide C (3) (25 mg) and papuamide D (4) (16 mg) as clear glasses.
Papuamide A (1). Amorphous glass; [R]25 ) +12.0° (c 3.47,
D
MeOH); UV [MeOH] λmax 227 (ꢀ 22 800), 274 (ꢀ 1400) nm; [MeOH
+ NaOH] λmax 233 (ꢀ 18 500) 285 (ꢀ 1900) nm; IR (neat, KCl) 3360,
2930, 1748, 1652, 1456, 1203 cm-1; HRFABMS of a CsI-doped sample
provided m/z 1548.6549 (M + Cs)+, calculated 1548.6602 for
1
C66H105N13O21Cs; H NMR see Table 1; 13C NMR see Table 1.
Papuamide B (2). Amorphous glass; [R]25 ) +12.9° (c 0.13,
D
MeOH); UV [MeOH] λmax 225 (ꢀ 24 000), 274 (ꢀ 1900) nm;
HRFABMS of a CsI-doped sample provided m/z 1534.6486 (M + Cs)+,
1
calculated 1534.6445 for C65H103N13O21Cs; H NMR see Table 2; 13C
NMR see Table 2.
Papuamide C (3). Clear glass; [R]25 ) +4.4° (c 1.14, MeOH);
D
UV [4:1 CH3CN-H2O] λmax 229 (ꢀ 18 470), 276 (ꢀ 2722) nm;
HRFABMS provided m/z 1399.7361 (M + H)+, calculated 1399.7361
for C66H103N12O21, and m/z 1421.7211 (M + Na)+, calculated 1421.7181
1
for C66H102N12O21Na; H NMR (500 MHz, 4:1 CD3CN-H2O) δ Hpr
H-2, 5.0 (1H); H-3, 1.61 (1H) and 2.07 (1H); H-4, 1.65 (2H); H-5,
1.30 (1H) and 1.65 (1H); H-6, 3.06 (1H) and 4.03 (1H); â-OMeTyr
H-R, 5.25 (1H); H-â, 4.22, (1H); OMe on C-â, 2.99 (3H); H-2 and
H-6, 7.17 (2H); H-3 and H-5, 6.74 (2H); NH, 7.67 (1H); NMeThr
H-2, 4.30 (1H); H-3, 3.82 (1H); H-4, 0.39 (3H); N-Me, 3.01 (3H); Ala
H-2, 4.44 (1H); H-3, 1.39 (3H); NH, 7.02 (1H); Gly 1 H-2, 3.44 (1H)
and 4.08 (1H); NH, 8.32 (1H); 3-OMeAla H-2, 4.11 (1H); H-3, 3.62
(1H) and 3.78 (1H); OMe on C-3, 3.29 (3H); NH, 7.96 (1H); 3-OHLeu
H-2, 5.00 (1H); H-3, 5.25 (1H); H-4, 1.90 (1H); H-5, 0.84 (3H); H-6,
0.77 (3H); NH 8.65 (1H); 3,4-DiMeGln H-2, 4.28 (1H); H-3, 2.07
(1H); Me on C-3, 0.95 (3H); H-4, 2.56 (1H); Me on C-4, 1.08 (3H);
NH on C-2, 8.07 (1H); Aba H-3, 6.72 (1H); H-4, 1.65 (3H); NH, 9.05
(1H); Thr H-2, 4.47 (1H); H-3, 4.29 (1H); H-4, 1.16 (3H); NH, 7.82
(1H); Gly 2 H-2, 3.78 (1H) and 3.98 (1H); NH, 8.32 (1H); Dhtda Me
on C-2, 1.19 (3H); H-3, 4.79 (1H); H-4, 5.46 (1H); H-5, 6.10 (1H);
Me on C-6, 1.73 (3H); H-7, 5.20 (1H); H-8, 2.31 (1H); Me on C-8,
0.89 (3H); H-9, 1.16 (1H) and 1.32 (1H); H-10, 0.77 (3H). Most protons
appeared as two resonances and in general were very broad and poorly
resolved; hence, only the chemical shift is given. Assignments and some
chemical shifts based on HMQC, HMBC, and COSY data. 13C NMR
(125 MHz, 4:1 CD3CN-H2O) δ Hpr CO, 170.8; C-2, 54.0; C-3, 26.6;
C-4, 21.1; C-5, 25.3; C-6, 44.6; â-OMeTyr CO, 171.8-172.8; C-R,
52.3; C-â, 84.8; OMe on C-â, 57.1; C-1, 129.1; C-2 and C-6, 130.8;
C-3 and C-5, 116.2; C-4, 158.1; NMeThr C-1, 169.3; C-2, 64.2; C-3,
64.0; C-4, 19.7; N-Me, 31.3; Ala C-1, 174.5; C-2, 48.4; C-3, 15.6;
Gly 1 C-1, 171.8-172.8; C-2, 42.8; 3-OMeAla C-1, 171.8-172.8;
C-2, 57.6; C-3, 71.2; OMe on C-3, 59.4; 3-OHLeu C-1, 171.8-172.8;
C-2, 55.9; C-3, 80.1; C-4, 28.8; C-5, 19.7; C-6, 18.7; 3,4-DiMeGln
C-1, 175.3; C-2, 58.9; C-3, 37.6; Me on C-3, 13.1; C-4, 41.1; Me on
C-4, 15.4; C-5, 179.6; Aba C-1, 166.8; C-2, 129.5; C-3, 135.2; C-4,
13.6; Thr C-1, 171.8-172.8; C-2, 59.9; C-3, 69.0; C-4, 19.9; Gly 2
C-1, 171.8-172.8; C-2, 43.8; Dhtda C-1, 178.6; C-2, 78.9; Me on
C-2, 22.4; C-3, 73.0; C-4, 125.0; C-5, 139.1; C-6, 131.8; Me on C-6,
16.9; C-7, 139.8; C-8, 35.0; Me on C-8, 20.9; C-9, 40.0; C-10, 12.5.
Preparation of Diacetate 5. A MeOH solution containing 7 mg of
papuamide A (1) was treated with 200 µL of acetic anhydride for 1 h.
Removal of the solvent under a stream of N2 provided the N-acetate
derivative which was dissolved in anhydrous pyridine and reacted with
200 µL of acetic anhydride. After 3 h the solvent was removed in vacuo
and the residue purified by C18 HPLC eluted with H2O for 10 min
followed by a gradient of increasing concentration of CH3CN in H2O
(30% CH3CN to 70% CH3CN over 15 min). Diacetate 5 was the
principal reaction product (2 mg), eluting with a retention time of 18.5
min. HRFABMS of a CsI-doped sample provided m/z 1632.6776 (M
+ Cs)+, calculated 1632.6813 for C70H109N13O23Cs; 1H NMR (500
MHz, 4:1 CD3CN-H2O) δ Hpr H-2, 5.02 (1H, d, J ) 3.9 Hz); H-3,
1.66 (1H, m) and 2.07 (1H, m); H-4, 1.15 (1H, m) and 1.69 (1H, m);
H-5, 1.35 (1H, m) and 1.66 (1H, m); H-6, 3.13 (1H, dt, J ) 14.2 and
5.4 Hz); â-OMeTyr H-R, 5.26 (1H, t, J ) 9.3 Hz); H-â, 4.34 (1H, d,
J ) 9.3 Hz); OMe on C-â, 3.03 (3H, s); NH, 7.71 (1H, d, J ) 9.3 Hz);
H-2 and H-6, 7.05 (2H, d, J ) 8.8 Hz); H-3 and H-5, 7.40 (2H, d, J )
8.8 Hz); OAc on C-4, 2.22 (3H, s); NMeThr H-2, 4.32 (1H, d, J )
6.8 Hz); H-3, 3.82 (1H, m); H-4, 0.40 (3H, d, J ) 6.8 Hz); N-Me, 3.01
(3H, s); Ala H-2, 4.47 (1H, quint, J ) 6.3 Hz), H-3, 1.40 (3H, d, J )
7.3 Hz); NH, 7.06 (1H, d, J ) 5.9 Hz); Gly 1 H-2, 3.78 (1H, dd, J )
7.8 and 4.9 Hz) and 4.13 (1H, dd, J ) 7.8 and 3.4 Hz); NH, 8.41 (1H,
dd, J ) 4.9 and 3.4 Hz); 3-OMeAla H-2, 4.14 (1H, dt, J ) 3.9 and
3.4 Hz); H-3, 3.69 (1H, dd, J ) 10.8 and 3.4 Hz) and 3.79 (1H, dd, J
) 10.8 and 3.4 Hz); OMe on C-3, 3.32 (3H, s); NH, 8.04 (1H, d, J )
3.9 Hz); 3-OHLeu H-2, 5.15 (1H, t, J ) 10.3 Hz); H-3, 5.32 (1H, dd,
J ) 10.3 and 2.0 Hz); H-4, 1.91 (1H, m); H-5, 0.79 (3H, d, J ) 6.4
Hz); H-6, 0.85 (3H, d, J ) 6.4 Hz); NH, 8.37 (1H, d, J ) 10.3 Hz);
3,4-DiMeGln H-2, 4.31 (1H, dd, J ) 5.4 and 3.4 Hz); H-3, 2.10 (1H,
m); Me on C-3, 0.98 (3H, d, J ) 6.8 Hz); H-4, 2.60 (1H, dq, J ) 7.3
and 3.4 Hz); Me on C-4, 1.14 (3H, d, J ) 7.3 Hz); C-2 NH, 8.58 (1H,
d, J ) 5.4 Hz); C-5 NH, 6.46 (1H, br s) and 6.95 (1H, br s); Dab H-2,

5908 J. Am. Chem. Soc., Vol. 121, No. 25, 1999
Ford et al.
4.39 (1H, dd, J ) 6.8 and 3.9 Hz); H-3, 4.27 (1H, m); H-4, 1.16 (3H,
d, J ) 7.3 Hz); C-2 NH, 7.67 (1H, d, J ) 7.3 Hz); C-3 NH, 7.58 (1H,
d, J ) 8.3 Hz); N-Ac on C-3, 1.85 (3H, s); Thr H-2, 4.38 (1H, dd, J
) 6.8 and 3.7 Hz); H-3, 4.20, (1H, dq, J ) 6.4 and 3.7 Hz); H-4, 1.08
(1H, d, J ) 6.4 Hz); NH, 8.12 (1H, d, J ) 6.8 Hz); Gly 2 H-2, 3.80
(1H, dd, J ) 8.3 and 5.9 Hz) and 4.09 (1H, dd, J ) 8.3 and 5.9 Hz);
NH, 8.27 (1H, t, J ) 5.9 Hz); Dhtda Me on C-2, 1.18 (3H, d, J ) 2.9
Hz); H-3, 4.79 (1H, d, J ) 10.3 Hz); H-4, 5.42 (1H, dt, J ) 10.3 and
2.9 Hz); H-5, 6.11 (1H, dt, J ) 11.7 and 3.4 Hz); Me on C-6, 1.74
(3H, d, J ) 3.4 Hz); H-7, 5.21 (1H, d, J ) 9.8 Hz); H-8, 2.31 (1H, m);
Me on C-8, 0.91 (3H, d, J ) 6.8 Hz); H-9, 1.20 (1H, m) and 1.33 (1H,
m); H-10, 0.81 (3H, t, J ) 7.8 Hz); 13C NMR (125 MHZ, 4:1 CD3-
CN-H2O), chemical shifts and assignments were deduced from HSQC
and HMBC correlations, δ Hpr CO, 170.8; C-2, 53.4; C-3, 26.4; C-4,
20.8; C-5, 25.4; C-6, 44.1; â-OMeTyr CO, 172.2; C-R, 51.9; C-â,
84.2; OMe on C-â, 57.2; C-1, 136.0; C-2 and C-6, 130.4; C-3 and
C-5, 122.7; C-4, 151.5; OAc on C-4, 171.2 and 20.7; NMeThr C-1,
169.4; C-2, 63.8; C-3, 63.3; C-4, 19.7; N-Me, 30.8; Ala C-1, 174.4;
C-2, 48.2; C-3, 15.1: Gly 1 C-1, 171.5; C-2, 42.9: 3-OMeAla C-1,
not observed; C-2, 57.3; C-3, 70.7; OMe on C-3, 59.1; 3-OHLeu C-1,
172.9; C-2, 55.5; C-3, 79.7; C-4, 28.5; C-5, 18.2; C-6, 19.2; 3,4-
DiMeGln C-1, 174.7; C-2, 58.2; C-3, 37.9; Me on C-3, 13.1; C-4,
41.4; Me on C-4, 15.1; C-5, 179.6; Dab C-1, 171.6; C-2, 59.0; C-3,
47.6; C-4, 16.1; Thr C-1, 172.3; C-2, 59.4; C-3, 68.1; C-4, 19.2; Gly
2 C-1, 172.1; C-2, 43.5; Dhtda C-1, 178.3; C-2, not observed; Me on
C-2, 21.8; C-3, 72.2; C-4, 125.3; C-5, 138.8; C-6, 131.8; Me on C-6,
16.6; C-7, 139.3; C-8, 34.7; Me on C-8, 20.2; C-9, 30.6; C-10, 12.0.
7.98 (1H, d, J ) 6.1 Hz); Dhab H-1, 3.30 (1H, dd, J ) 10.7 and 6.7
Hz) and 3.42 (1H, dd, J ) 10.7 and 6.7 Hz); H-2, 3.62 (1H, m); H-3,
3.82 (1H, m); H-4, 0.96 (3H, d, J ) 6.3 Hz); NH, 7.29 (1H, d, J ) 9.3
Hz); 13C NMR (125 MHZ, DMSO-d6) δ Dhtda C-1, 175.4; C-2, 76.7;
C-3, 70.8; C-4, 126.9; C-5, 135.7; C-6, 130.6; C-7, 137.5; C-8, 33.5;
C-9, 29.8; C-10, 11.9; Gly C-1, 168.9; C-2, 42.2; Dhab C-1, 60.4;
C-2, 55.8; C-3, 64.4; C-4, 20.1; Many protons and carbons appeared
as two resonances, in which case only the signal for the major one
(>90% excess) is reported above. Assignments based on HMQC,
HMBC, COSY, ROESY, and NOE data.
Interconversion of Papuamide A (1) to Papuamide C (3) by
Hofmann Elimination. To 6.8 mg of papuamide A (1) was added 3
mL of freshly distilled DMF and 0.5 mg of K2CO3. The solution was
stirred for 2 h, and then 100 µL of MeI (passed over dry basic alumina)
was added and the reaction stirred for a further 15 h at room
temperature. The volume of the solution was reduced in vacuo to 0.25
mL. The resulting solution was fractionated by reversed-phase C18
HPLC with 39:61 CH3CN-0.05% aqueous TFA as eluent to yield one
major product (5.7 mg) that was identical to papuamide C (3) by co-
injection on HPLC (a single sharp peak was observed by analytical
HPLC), 1H NMR, and positive ion HRFABMS (M + Na)+ m/z
1421.7183 (C66H102N12O21Na, calculated 1421.7181).
Synthesis of N-Methylthreonine Standards. The synthetic method
described by Beulshausen et al.22 was modified as described below.
SOCl2 (0.26 mL) was added dropwise to 1 mL of dry MeOH, and the
solution was cooled to -10 °C. To this solution was added 100 mg of
L-threonine (2S,3S-threonine), and the mixture was stirred at room
temperature for 12 h. The reaction mixture was evaporated to dryness,
and the resulting threonine methyl ester (Thr-OMe) was crystallized
from petroleum ether. To a solution of Thr-OMe in CH2Cl2 (10 mL)
was added 0.1 N aqueous trifluoroacetic acid (TFA) (10 mL), and the
mixture was cooled to 0 °C. With vigorous stirring, a 37% aqueous
solution of formaldehyde (62 µL) was added dropwise, and stirring
was continued at room temperature for 8 h. The reaction mixture was
neutralized with NaHCO3 and the product isolated by extraction with
CH2Cl2 (4 × 10 mL). The combined organic extracts were dried with
MgSO4 and concentrated in vacuo to give the oxazolidine derivative,
which was used without further purification. A solution of the
oxazolidine in CH2Cl2 (6 mL) was cooled to 0 °C, and TFA (6 mL)
and triethylsilane (0.6 mL) were added. The mixture was stirred at room
temperature for 24 h, then the solvent was removed in vacuo, and the
residue was dissolved in 1 N HCl and washed with petroleum ether.
Hydrolysis in 6 N HCl (400 µL) at 110 °C for 16 h provided (2S,3S)-
N-MeThr. The identical procedure using L-allo-threonine, D-threonine,
or D-allo-threonine as starting material provided (2S,3R)-, (2R,3S)-, or
(2R,3R)-N-MeThr, respectively.
Hydrolysis of Papuamide A (1) with Triethylamine. A solution
of papuamide A (1) (20.3 mg) in 2 mL of 5% aqueous triethylamine
was heated at 49 °C for 16 h with stirring. The solution was evaporated
to dryness in vacuo and the reaction mixture chromatographed by C18
reversed-phase HPLC using a linear gradient of (A) 0.05% aqueous
TFA and (B) CH3CN with 0% B at start to 100% B over 120 min to
give pure tripeptide 6 (3.2 mg). Tripeptide 6: isolated as a clear oil;
[R]25D ) -73.2° (c 0.21, MeOH); UV [CH3CN-H2O] λmax 240 (ꢀ 675),
276 (ꢀ 654) nm; HRFABMS provided m/z 438.2235 (M + H)+,
calculated 438.2240 for C21H32N3O7 and m/z 460.2068 (M + Na)+,
calculated 460.2060 for C21H31N3O7Na; 1H NMR (500 MHZ, DMSO-
d6) δ Hpr H-2, 5.14 (1H, br d, J ) 5.0 Hz); H-3, 1.55 (1H, m) and
2.15 (1H, br d, J ) 13.0 Hz); H-4, 1.26 (1H, m) and 1.67 (1H, m);
H-5, 1.42 (1H, m) and 1.67 (1H, m); H-6, 3.12 (1H, t, J ) 12.3 Hz)
and 4.21 (1H, br d, J ) 12.3 Hz); â-OMeTyr H-R, 5.19 (1H, t, J )
9.7 Hz); H-â, 4.31 (1H, d, J ) 9.7 Hz); H-2 and H-6, 7.14 (2H, d, J
) 8.0 Hz); H-3 and H-5, 6.70 (2H, d, J ) 8.0 Hz); OMe on C-â, 3.00
(3H, s); NH, 9.17 (1H, d, J ) 9.7 Hz); NMeThr H-2, 3.27 (1H, m);
H-3, 3.42 (1H, m); H-4, 0.32 (3H, d, J ) 6.0 Hz); N-Me, 2.28 (3H,
m); NH, 8.55 (1H, br s); NH′, 8.95 (1H, br s); 13C NMR (125 MHz,
DMSO-d6) δ Hpr CO, 172.0; C-2, 51.5; C-3, 26.2; C-4, 20.4; C-5,
24.7; C-6, 43.3; â-OMeTyr CO, 169.6; C-R, 51.7; C-â, 82.6: OMe
on C-â, 56.1; C-1, 127.4; C-2 and C-6, 129.2; C-3 and C-5, 114.9;
C-4, 157.5; NMeThr C-1, 164.5; C-2, 66.7; C-3, 65.0; C-4, 18.8; N-Me,
31.2. Many protons and carbons appeared as two resonances, in which
case only the signal for the major one (>90% excess) is reported above.
Assignments based on HMQC, HMBC, COSY, ROESY, and NOE data.
Determination of Absolute Stereochemistry. (a) Peptide Hy-
drolysis. Peptide samples (200 µg) were dissolved in degassed 6 N
HCl (0.5 mL) in an evacuated glass tube and heated at 108 °C for 16
h. The solvent was removed in vacuo and the residue placed under
high vacuum.
(b) LC-MS Analysis of Marfey’s (FDAA) Derivatives.23 An
aqueous solution of the hydrolysate (20 µL) was treated with 6%
triethylamine (10 µL) and 1% 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide in acetone (20 µL) at 40 °C for 1 h. The reaction mixture was
diluted with 50 µL of H2O and an aliquot applied to a C18 HPLC column
eluted with CH3CN-0.05% aqueous TFA using a linear gradient from
10% CH3CN to 50% CH3CN over 20 min. Derivatized amino acids
were detected by absorption at 340 nm and by MS (mass range 300-
800 Da). Retention times (min) are given in parentheses: L-Thr (9.94),
L-allo-Thr (10.46), N-Me-L-Thr (11.07), D-allo-Thr (11.78), N-Me-D-
Thr (12.07), OMe-L-Ala (12.50), L-Ala (12.86), d-Thr (13.47), N-Me-
L-allo-Thr (13.89), N-Me-D-allo-Thr (14.60), OMe-D-Ala (15.76), D-Ala
(15.82), D-Hpr (18.46), L-Hpr (19.74).
(c) LC-MS Analysis of 2,3,4,6-Tetra-O-acetyl-â-D-glucopyran-
osyl Isothiocyanate (GITC) Derivatives. An aqueous solution of the
hydrolysate (20 µL) was treated with 6% triethylamine (10 µL) and
1% 2,3,4,6-tetra-O-acetyl-â-D-glucopyranosyl isothiocyanate in acetone
(20 µL) at room temperature for 5 min. The reaction mixture was
applied directly to a C18 HPLC column eluted as described above for
the FDAA derivatives. Derivatized amino acids were detected by
Sodium Borohydride Reduction of Papuamide A (1). A solution
of papuamide A (1) (5.4 mg) in 2 mL of 70% aqueous 2-propanol and
excess NaBH4 (19.8 mg) was stirred for 16 h at room temperature.
The solution was evaporated to dryness in vacuo and the reaction
mixture chromatographed in a manner identical to that described above
for the purification of 6, to give both the tripeptide 6 (0.5 mg) (retention
time 40.3 min) and the tetraol 7 (0.1 mg) (retention time 48.7 min).
Tetraol 7: isolated as a clear oil; UV [CH3CN-H2O] λmax 236 (ꢀ 9030)
nm; HRFABMS provided m/z 409.2324 (M + Na)+, calculated
409.2315 for C19H34N2O6Na, m/z 391.2215 (M + Na - H2O)+,
calculated 391.2209 for C19H32N2O5Na, and m/z 369.2403 (M + H -
1
2H2O)+, calculated 369.2389 for C19H31N2O4; H NMR (500 MHZ,
DMSO-d6) δ Dhtda Me on C-2, 1.08 (3H, s); H-3, 4.60 (1H, d, J )
10.3 Hz); H-4, 5.39 (1H, m); H-5, 5.98 (1H, d, J ) 12.3 Hz); Me on
C-6, 1.73 (3H, dd, J ) 4.1 and 1.1 Hz); H-7, 5.22 (1H, m); H-8, 2.32
(1H, m); Me on C-8, 0.91 (3H, d, J ) 6.7 Hz); H-9, 1.21 (1H, m) and
1.33 (1H, m); H-10, 0.82 (3H, m); Gly H-2, 3.65-3.75 (2H, m); NH,

Papuamides A-D
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5909
absorption at 254 nm and by MS (mass range 400-950). Retention
times (min) are given in parentheses: L-allo-Thr (12.08), L-Thr (12.40),
N-Me-L-allo-Thr (12.45), D-allo-Thr (12.53), L-Ala (12.60), N-Me-L-
Thr (12.76), N-Me-D-allo-Thr (13.17), D-Thr (13.52), OMe-L-Ala
(13.54), D-Ala (13.67), N-Me-D-Thr (14.01), OMe-D-Ala (14.44), L-Hpr
(15.79), D-Hpr (16.83).
We also thank Mike Leblanc for assisting in the collection of
T. swinhoei. We express gratitude for the help and services
provided by Dr. Larry Orsak, Director of the Christensen
Research Institute, Madang, Papua New Guinea. We also thank
Dr. Michael Currens for anti-HIV bioassays. Stereospecific
synthetic intermediates of 2,3-diaminobutyric acid and an
authentic sample of aciculitin B were kindly provided by Dr.
D. John Faulkner. We thank the laboratory of the late Dr. Luigi
Minale for kindly providing a sample of callipeltin A. Enan-
tiomerically pure forms of 3-hydroxyleucine were kindly
provided by Dr. Amos B. Smith, III. Work at the University of
British Columbia was supported by NIH Grant CA 67786. This
paper constitutes number 58 in the NCI Laboratory of Drug
Discovery Research and Development series “HIV-Inhibitory
Natural Products”; for part 57 in the series, see ref 27.
(d) GC-MS Analysis of Trifluoroacetyl Isopropyl Ester Deriva-
tives. A premixed solution of acetyl chloride-isopropyl alcohol (1:4)
was added to the hydrolysate and heated to 100 °C for 1 h. The solution
was dried under a stream of N2, and trifluoroacetic anhydride (50 µL)
in CH2Cl2 (200 µL) was added to the residue. The reaction mixture
was heated to 100 °C for 20 min, then cooled to 0 °C, and evaporated
under a stream of N2. The product was taken up in ethyl acetate and
immediately analyzed by GC-MS using a Chirasil-L-Val capillary
column ramped from 40 to 240 °C at 10 °C/min, and a mass range of
40-650 Da was recorded every 1 s. Retention times (min) are given
in parentheses: (2R,3S)-OHLeu (8.97), (2S,3R)-OHLeu (9.17), OMe-
d-Ala (9.26), NMe-L-Thr (9.37), D-Ser (9.93), (2R,3R)-OHLeu (10.87),
(2S,3S)-OHLeu (11.18), L-Hpr (11.42), (2S,3S,4R)-diMePyroGlu (12.77),
(2R,3S)-Dab (14.48), (2S,3S,4R)-diMeGlu (14.74), (2S,3R)-Dab (14.80),
(2R,3R)-Dab (15.60), (2S, 3S)-Dab (16.18).
Supporting Information Available: NMR spectral data for
papuamide A (1), papuamide B (2), and the diacetate derivative
1
of papuamide A (5), including H NMR, 13C NMR, HSCQ,
HMBC, TOCSY, and ROESY spectra (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank the Coral Reef Foundation and
Dr. Patrick Colin for collecting the specimen of T. mirabilis.
JA990582O