Gymnangiamide, a Cytotoxic Pentapeptide from the Marine Hydroid Gymnangium regae

Article abstract of DOI:10.1021/jo0303113

A cytotoxic aqueous extract from the marine hydroid Gymnangium regae provided a novel linear pentapeptide, designated gymnangiamide (1). The planar structure of 1 was elucidated by interpretation of spectral data as well as chemical degradation and derivatization studies. In addition to the amino acids isoleucine and phenylserine, this peptide contained N-desmethyldolaisoleuine, O-desmethyldolaproine, and α-guanidino serine, three residues that have not previously been reported in a natural product. The absolute configurations of the constituent amino/guanidino acids were determined by chemical degradation and derivatization, followed by HPLC and LC-MS comparison with authentic standards. Gymnangiamide (1) was moderately cytotoxic against a number of human tumor cell lines in vitro.

Full text of DOI:10.1021/jo0303113

Gym n a n gia m id e, a Cytotoxic P en ta p ep tid e fr om th e Ma r in e
Hyd r oid Gym n a n giu m r ega e
,|
Dennis J . Milanowski,† Kirk R. Gustafson,*,† Mohammad A. Rashid,‡,§ Lewis K. Pannell,
J ames B. McMahon,† and Michael R. Boyd#
Molecular Targets Development Program, Center for Cancer Research, National Cancer Institute,
Building 1052, Room 121, Frederick, Maryland 21702-1201, Intramural Research Support Program,
SAIC-Frederick, Frederick, Maryland 21702-1201, Laboratory of Bioorganic Chemistry, National Institute
of Diabetes and Digestive and Kidney Disease, Bethesda, Maryland 20892-0805, and USA Cancer
Research Institute, College of Medicine, University of South Alabama, Mobile, Alabama 36688
manuscripts@mail.ncifcrf.gov
Received October 10, 2003
A cytotoxic aqueous extract from the marine hydroid Gymnangium regae provided a novel linear
pentapeptide, designated gymnangiamide (1). The planar structure of 1 was elucidated by
interpretation of spectral data as well as chemical degradation and derivatization studies. In addition
to the amino acids isoleucine and phenylserine, this peptide contained N-desmethyldolaisoleuine,
O-desmethyldolaproine, and R-guanidino serine, three residues that have not previously been
reported in a natural product. The absolute configurations of the constituent amino/guanidino acids
were determined by chemical degradation and derivatization, followed by HPLC and LC-MS
comparison with authentic standards. Gymnangiamide (1) was moderately cytotoxic against a
number of human tumor cell lines in vitro.
In tr od u ction
dithiocarbamate14 and piperidinol15 containing metabo-
lites with antifeedant activity. It has been suggested that
the relatively modest amount of chemical data published
on hydroids is due to the difficulty of collection and
identification, and not due to a lack of chemical diversity
within these organisms.2,9
An aqueous extract of the marine hydroid Gym-
nangium regae J aderholm, collected in the Philippines,
was selected for chemical analysis based on the pattern
of differential cytotoxicity it produced in the U.S. Na-
tional Cancer Institue (NCI)’s 60 cell line antitumor
screen.16,17 Cytotoxicity-guided fractionation of the extract
led to the isolation of a novel cytotoxic linear pentapep-
tide, which has been named gymnangiamide (1). Of the
five residues that comprise this peptide, two were un-
usual amino acids that are reported for the first time
from a natural source, and one is a rare R-guanidino acid
that has not previously been found in a naturally
occurring peptide. This report represents the first chemi-
cal investigation of the genus Gymnangium and the first
description of a peptide from a hydroid.
Cnidarians have been the subject of extensive chemical
study resulting in the isolation of a large number of
unique secondary metabolites. Most of these studies have
focused on soft corals and gorgonians, while relatively
little attention has been given to marine hydroids
(class: Hydrozoa). Compounds that have been reported
from marine hydroids include steroids1-4 and other
terpenoids,5 fatty acid derivatives,6,7 antimicrobial and
cytotoxic aromatic polyketides,8-12 â-carbolines,13 and
* To whom correspondence should be addressed. Phone: (301) 846-
5391. Fax: (301) 846-6919.
† Molecular Targets Development Program, CCR, NCI.
‡ SAIC-Frederick.
§ On leave from the Department of Pharmacy, University of Dhaka,
Dhaka-1000, Bangladesh.
Laboratory of Bioorganic Chemistry, NIDDK.
| Present address: USA Cancer Research Institute, College of
Medicine, University of South Alabama, Mobile, AL 36688.
#
USA Cancer Research Institute.
(1) Cimino, G.; De Rosa, S.; De Stefano, S.; Sodano, G. Tetrahedron
Lett. 1980, 21, 3303-3304.
(2) Fattorusso, E.; Lanzotti, V.; Magno, S.; Novellino, E. J . Org.
Chem. 1985, 50, 2868-2870.
(3) Aiello, A.; Fattorusso, E.; Magno, S. J . Nat. Prod. 1987, 50, 191-
(10) Fahy, E.; Andersen, R. J .; Xu, C.; Clardy, J . J . Org. Chem. 1986,
51, 5145-5148.
194.
(4) De Rosa, S.; Milone, A.; Popov, S.; Andreev, St. Comp. Biochem.
Physiol. 1999, 123B, 229-233.
(11) Fahy, E.; Andersen, R. J . Can. J . Chem. 1987, 65, 376-383.
(12) Pathirana, C.; Andersen, R. J .; Wright, J . L. C. Can. J . Chem.
1990, 68, 394-396.
(5) Kato, T.; Kumanireng, A. S.; Ichinose, I.; Kitahara, Y.; Kaki-
numa, Y.; Nishihira, M.; Kato, M. Experientia 1975, 31, 433-434.
(6) Seo, Y.; Cho, K. W.; Rho, J .-R.; Shin, J .; Kwon, B.-M.; Bok, S.
H.; Song, J .-I. Tetrahedron 1996, 52, 10583-10596.
(7) Fusetani, N.; Yasukawa, K.; Matsunaga, S.; Hashimoto, K.
Comp. Biochem. Physiol. 1986, 83B, 511-513.
(8) Fahy, E.; Andersen, R. J .; Cun-heng, H.; Clardy, J . J . Org. Chem.
1985, 50, 1149-1150.
(13) Aiello, A.; Fattorusso, E.; Magno, S.; Mayol, L. Tetrahedron
1987, 43, 5929-5932.
(14) Lindquist, N.; Lobkovsky, E.; Clardy, J . Tetrahedron Lett. 1996,
37, 9131-9134.
(15) Lindquist, N.; Shigematsu, N.; Pannell, L. J . Nat. Prod. 2000,
63, 1290-1291.
(16) Boyd, M. R. Current Therapy in Oncology; Niederhuber, J . E.,
Ed.; B. C. Decker, Inc.: Philadelphia, PA, 1993; pp 11-12.
(17) Boyd, M. R. Drug Dev. Res. 1995, 34, 91-109.
(9) Fahy, E.; Andersen, R. J .; Van Duyne, G. D.; Clardy, J . J . Org.
Chem. 1986, 51, 57-61.
10.1021/jo0303113 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/26/2004
3036
J . Org. Chem. 2004, 69, 3036-3042

Gymnangiamide
amino acids, N-desmethyldolaisoleuine (Ddil) and O-
desmethyldolaproine (Ddap), and a single guanidino acid,
R-guanidino serine (Gser). The structures of the unusual
residues were assigned as follows. The R-methylene of
Ddil was established by HMBC correlations between the
methylene protons (δ 2.26, 2.18) and the carbonyl at δ
171.62 and methine carbons at δ 54.37 and δ 78.79.
COSY correlations were observed between the R-meth-
ylenes and the â-oxymethine (δ 4.06), which in turn
coupled to the γ-methine proton (δ 4.09), which also
correlated with the amide proton at δ 8.28. This estab-
lished the γ-amino acid Ddil backbone. The side chain
was assigned on the basis of 1D-TOCSY, COSY, and
HMBC data, while a methoxyl group was placed at the
â position in Ddil by an HMBC correlation between the
methyl protons (δ 3.28) and the â-oxymethine carbon. The
Ddap residue was similarly defined by COSY correlations
from the R-methine (δ 2.36) to a methyl group (δ 1.24)
and the â-oxymethine (δ 3.79), which in turn was coupled
to the γ-methine (δ 3.22). The heterocyclic ring of Ddap
was defined by 1D-TOCSY, COSY, and HMBC correla-
tions. These unusual residues are closely related to
residues found in the dolastatin family of peptides except
that in both there is reduced methylation; Ddil lacks the
N-methyl group found in dolaisoleuine (Dil), and Ddap
lacks the â-methoxy group found in dolaproine (Dap).18-20
To the best of our knowledge, this is the first isolation of
these desmethyl amino acid derivatives from a natural
source.
Resu lts a n d Discu ssion
A 12.8-g portion of the aqueous extract from Gym-
nangium regae was dissolved in H2O, applied to a wide-
pore C4 vacuum liquid chromatography column, and
sequentially eluted with 100% H2O, H2O-MeOH (2:1),
H2O-MeOH (1:2), and 100% MeOH. The H2O-MeOH
(1:2) fraction was chromatographed on Sephadex LH-20
eluted with MeOH-H2O (7:3) and final purification by
gradient elution C18 HPLC with increasing concentra-
tions of MeCN in H2O provided 2.4 mg of gymnangiamide
(1). The positive ion FABMS of 1 exhibited pseudo-
molecular ions at m/z 750.5 for [M + H]+ and m/z 772.5
for [M + Na]+, which indicated a nominal molecular
weight of 749. The molecular formula was established
as C36H59N7O10 by high-resolution FABMS of a CsI-
doped sample ([M + Cs]+ m/z 882.3374, calcd for
C36H59N7O10Cs 882.3377) in combination with a detailed
The final residue in 1 was the unusual guanidino acid,
R-guanidino serine. HMBC and COSY correlations from
the R-methine proton (δ 4.24) to the primary alcohol
group (δ 3.81, 3.85) established the presence of a serine
moiety. HMBC correlations from both the R-methine
proton and the NH proton doublet at δ 7.45 to a carbon
at δ 159.2 suggested the presence of a guanidine func-
tionality. Support for this assignment was provided by
treatment of the peptide on TLC with either FCNP
(ferricyante-sodium nitroprusside) or Sakaguchi spray
reagents21 which both tested positive for the presence of
guanidine. Guanidino analogues of common amino acids
are quite rare in nature; however, unusual R-guanidino
acids have been encountered as constituents of phega-
nomycin and other related antibiotic peptides isolated
from terrestrial fungi.22,23 Gymnangiamide (1) represents
the first marine peptide found to contain an R-guanidino
acid residue. The five residues established above ac-
counted for all of the unsaturations present in 1, indicat-
ing that it was a linear peptide with a free C-terminus
and a guanidine at the N-terminus. This was confirmed
by formation of the carboxymethyl ester 2 on treatment
with (trimethylsilyl)diazomethane24 and formation of the
1
analysis of the H and 13C NMR data. A cursory exami-
nation of the 1H and 13C NMR spectra indicated that
there were approximately twice as many signals present
as could be accounted for by the molecular formula.
Changing either the solvent composition or temperature
of the sample altered the ratio of the signals present,
indicating that there were multiple conformers of 1 in
solution, similar to that observed for other peptides.18,19
No experimental conditions were found which could
effectively coalesce the NMR signals into a single set of
resonances. The data reported below and in Table 1 are
for the most abundant form of 1.
The presence of 5 carbonyl resonances in the 13C NMR
spectrum, and 4 amide NH doublets (δ 7.45-8.28)
together with a number of upfield methyl doublets and
triplets in the 1H NMR spectrum of 1 (Table 1) suggested
it was a peptide. Extensive 1D and 2D NMR analyses of
1 established the presence of the amino acids isoleucine
(Ile) and phenyserine (Pser) together with the unusual
(20) Sone, H.; Shibata, T.; Fujita, T.; Ojika, M.; Yamada, K. J . Am.
Chem. Soc. 1996, 118, 1874-1880.
(21) Krebs, K. G.; Heusser, D.; Wimmer, H. Spray Reagents. In
Thin-Layer Chromatography; Stahl, E., Ed.; Springer-Verlag: New
York, 1969; pp 854-909.
(22) Leclercq, P. A.; Smith, L. C.; Desiderio, D. M., J r. Biochem.
Biophys. Res. Commun. 1971, 45, 937-944.
(18) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.;
Boetner, F. E.; Kizu, H.; Schmidt, J . M.; Baczynskyj, L.; Tomer, K. B.;
Bontems, R. J . J . Am. Chem. Soc. 1987, 109, 6883-6885.
(19) Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle,
D. G.; Paul, V. J .; Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A. J .
Nat. Prod. 1998, 61, 1075-1077.
(23) Takita, T.; Shimada, N.; Yagisawa, N.; Kato, K.; Naganawa,
H.; Hiroshi, K.; Umezawa, H. Proc. 15th Symp. Pept. Chem. 1977, 121-
126.
(24) Kondo, E.; Katayama, T.; Kawamura, Y.; Yasuda, Y.; Matsu-
moto, K.; Ishii, K.; Tanimoto, T.; Hinoo, H.; Kato, T.; Kyotani, H.; Shoji,
J . J . Antibiot. 1989, 42, 1-6.
J . Org. Chem, Vol. 69, No. 9, 2004 3037

Milanowski et al.
ROESY
TABLE 1. NMR Sp ectr a l Da ta for th e Gym n a n gia m id e (1) in CD3OHa
13Cb
1Hc
mult (J , Hz)
TOCSYd
HMBCd
phenylserine (Pser)
1
173.26 (s)
2
3
4
59.31 (d) 4.86
73.15 (d) 5.40
142.86 (s)
m
d (2.2)
H3, NH
H2, NH
C1, C3, C10
C4, C5,9
H3, H5,9
H2, H5,9
5,9
6,8
7
126.73 (d) 7.41, 2H bd (7.7)
129.09 (d) 7.30, 2H bd (7.7)
H6,8, H7
H5,9, H7
H5,9, H6,8
H2, H3
C3, C7
C4, C6,8
C5,9
H2, H3
128.16 (d) 7.22
8.25
m
d (9.2)
NH
C10
H2, H11
O-desmethyldolaproine (Ddap)
10
11
12
13
14
178.02 (s)
C10
C12, C17
C11, C13, C14, C17
45.15 (d) 2.36
74.80 (d) 3.79
60.88 (d) 3.22
26.10 (t)
24.30 (t)
48.27 (t)
m
m
m
m
m
m
m
m
H12, H17
H14b, H17, NH (Pser)
H13, H17, H19a
H12, H17, H19ab
H11, H13, H14ab, H17
H12, H14ab, H15ab, H16, H17
H13, H15b, H16
H13, H15a, H16, H17
H13, H14ab, H16
H13, H14ab, H16, H17
H13, H14ab, H15ab, H17
H11, H12, H13, H14b, H15b, H16
1.24
1.77
1.43
1.81
3.12
C12, C15
C14, C16
C14
C14, C15
C10, C11, C12
H11, H15a, H16
15
16
17
H14b
H11, H12, H13
16.29 (q) 1.24, 3H d (6.6)
N-desmethyldolaisoleuine (Ddil)
18
19
171.62 (s)
35.44 (t)
2.26
2.18
dd (∼1, 15.9) H20, H21, H22, NH
dd (8.8, 15.9) H20, H21, H22, NH
C18
C18, C20
C22
H12, H13, H20, H22a
H13
H25, H26
20
21
22
23
78.79 (d) 4.06
54.37 (d) 4.09
36.80 (d) 1.44
m
m
m
m
m
m
H19ab, H21, H22, H23ab, H25
H19ab, H20, H22, H23ab, H25, NH C19, C20, C22, C23, C27 H22, H25, H26, NH
H21, H23ab, H24, H25, NH
H21, H22, H24
H19a, H21, H22
C23
26.74 (t)
1.58
1.14
C22, C24, C25
C22, C24, C25
C22, C23
C21, C22, C23
C20
24
25
26
NH
10.98 (q) 0.84, 3H
16.18 (q) 0.92, 3H d (7.0)
57.30 (d) 3.28, 3H
8.28
H21, H22, H23ab
H21, H22, H23ab, NH
H20
H20
H28
s
d (9.9)
H19ab, H21, H22, H23ab, H25
isoleucine (Ile)
C27
27
28
29
30
174.43 (s)
59.78 (d) 4.29
38.05 (d) 1.83
m
m
m
m
m
H29, H30ab, H31, H32, NH
H28, H30ab, H31, H32, NH
H28, H29, H31, H32, NH
H28, H29, H31, H32, NH
H28, H29, H30ab, H32, NH
H28, H29, H30ab, H31, NH
H28, H29, H30ab, H31, H32
C27, C29, C30, C32, C33 H29, H32, NH (Ddil)
H28, H30b, H32
C29, C31, C32
25.79 (t)
1.23
1.59
C29, C31, C32
C29, C30
H29
H30b
31
32
NH
11.31 (q) 0.87, 3H
16.35 (q) 1.03, 3H d (6.6)
C28, C29, C30
C33
H28, H29
H29, H34
7.93
d (8.0)
R-guanidio serine (Gser)
33
34
35
170.03 (s)
58.38 (d) 4.24
m
m
H35ab, NH
H34, NH
dd (4.4, 10.6) H34, NH
C33, C35, C36
C33, C34
H35ab, NH (Ile)
H34
H34
63.92 (t)
3.81
3.85
36
159.17 (s)
NH
7.45
d (7.7) H34, H35ab
C35, C36
a
Spectra recorded at 500 MHz for 1H and 125 MHz for 13C and referenced to the residual solvent signal (δH 3.30, δC 49.0); only data
for the most abundant conformer are reported. Multiplicities determined by using the DEPT pulse sequence. c With geminal protons
the upfield resonance is designated “a” and the downfield resonance “b”. Geminal couplings are not shown.
b
d
dimethylpyrimidine derivative 3 (m/z 828.5 [M + H]+)
on subsequent treatment of 2 with 2,4-pentanedione.25
The linear sequence of 1 was established by a combi-
nation of NMR (Figure 1) and MS/MS methods (Figure
2). The R-methine proton (δ 4.24) of the Gser residue
showed a correlation to the carbonyl at δ 170.03 as did
the amide proton (δ 7.93) of Ile, which placed these two
F IGURE 1. Key HMBC (solid arrows) and ROESY (dashed
arrows) correlations for 1.
residues in sequence at the N-terminus. Similarly, the
amide proton (δ 8.28) of Ddil showed an HMBC correla-
sequence. The Ddap residue was connected to Ddil based
on a ROESY correlation from the â (δ 3.79) and γ (δ 3.22)
protons of the former to the R protons (δ 2.18, 2.26) of
the Ddil. Finally, Pser was placed at the C-terminus by
tion to the Ile carbonyl at δ 174.43 placing it next in the
(25) Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull.
1981, 29, 1475-1478.
3038 J . Org. Chem., Vol. 69, No. 9, 2004

Gymnangiamide
The four stereoisomers of isoleucine were readily resolved
by ligand-exchange chromatography on a (D)-penicil-
lamine column eluted with 2 mM Cu2+ and thus the
isoleucine residue in 1 was determined to be L-Ile
(28S,29S using the numbering scheme for 1).
Biosynthetic considerations suggested that Ddap might
have the same stereochemisty as the analagous residue
Dap, found in the dolastatins. Thus, the Ddap residue
in 1 was methylated to allow direct comparison with an
authentic sample of Dap. The intact peptide 1 was
methylated with NaH/MeI, hydrolyzed, and treated with
either FDAA or GITC. Both the FDAA and GITC deriva-
tives of the methylated Ddap from 1 had identical
retention times by LC-MS with similarly derivatized
authentic Dap, which allowed assignment of the stereo-
chemistry of Ddap as 11R,12R,13S. Under these condi-
tions Ddil did not methylate so 1 was first hydrolyzed,
and the free amino acids were acetylated and then
methylated as described above. A shortened acid hy-
drolysis removed the acetate and the product was then
derivatized with either FDAA or GITC. LC-MS of me-
thylated Ddil derivatized with either FDAA or GITC
showed retention times identical with that of authentic
Dil and established the stereochemistry of Ddil as
20R,21S,22S.
Of the four possible phenylserine stereoisomers, the
only standard available was a commercial mixture of the
D- and L-threo diastereomers. Acid-catalyzed racemization
of the commercial mixture by extended heating in HCl
provided the D- and L-erythro isomers. Initial results
showed that the FDAA derivatives of the four Pser
diastereomers could be clearly resolved by LC-MS and
that the isomer present in the hydrolysate of 1 cor-
responded to one of the threo isomers. The commercial
DL-threo-Pser mixture was derivatized with FMOC-Cl
and the FMOC protected D- and L-threo diastereomers
were separated by chiral HPLC on a Sumipax OA-3100
column. Deprotection and cleanup of the resulting frac-
tions provided the individual D- and L-threo compounds
which were identified by their optical rotations. Com-
parison of the acid hydrolyzate of 1 with the FDAA
derivatives of the individual threo-Pser isomers indicated
that L-threo-Pser (2S,3R) is present in 1.
Gymnangiamide (1) was tested for cytotoxic activity
by using a panel of 10 human tumor cell lines (see the
Experimental Section). Significant differential growth
inhibition, with IC50 values that ranged from 0.46 to >11
µg/mL, was observed, which was consistent with other
cytotoxins that are known to target tubulin. Gymnan-
giamide (1) is structurally related to the potent tubulin
interacting pentapeptide dolastatin 10 and its congeners;
however, it is significantly less cytotoxic than the
dolastatins.18-20,29,30 The dolastatins that have been
isolated to date are either cyclic or have their N-termini,
and often their C-termini blocked. The N-termini of linear
dolastatins are usually found as N,N-dimethyl deriva-
tives while the C-termini are variously modified and do
not typically exist as the free acid. Compound 1, with an
N-terminal guanidine moiety and a C-terminal carboxylic
acid, would exist as a zwitterion in cell culture media.
The ionic character of 1 could result in reduced uptake
by the cells and therefore reduced cytotoxicity. A com-
parative study showed that the C-terminally blocked
derivative 2 and the C- and N-terminally blocked deriva-
F IGURE 2. Positive ion FAB MS/MS fragmentation pattern
for 1.
an HMBC correlation from the Pser amide proton (δ 8.25)
to the Ddap carbonyl at δ 178.02. The linear sequence
was confirmed by MS/MS fragmentation analysis as
shown in Figure 2.
Unambiguous assignment of the absolute stereochem-
isty of 1 required different procedures for each of the
constituent guanidino or amino acids. Guanidino serine
was not detected in the acid hydrolysate of 1 after
derivatization of the hydrolysate with either N-(3-fluoro-
4,6-dinitrophenyl)-L-alaninamide (FDAA; Marfey’s re-
agent)26 or 2,3,4,6-tetra-O-acetyl-â-D-glucopyranosyl iso-
thiocyanate (GITC).27 Derivatization of a synthetic stan-
dard of Gser also failed to provide an observable deriva-
tive. This was overcome by treating the peptide with
hydrazine hydrate, either before or after complete acid
hydrolysis, to convert the guanidine group to a free amine
and thereby allow direct comparison with authentic
standards of D- and L-Ser.28 When 1 was treated with
hydrazine prior to acid hydrolysis there was good recov-
ery of Ser but complete racemization was observed. When
acid hydrolysis of 1 was performed first, the yield was
reduced but L-Ser was recovered in greater than 10:1
excess over D-Ser. Although acid hydrolysis of the intact
peptide prior to cleavage of the guanidine moiety resulted
in a significant loss of signal, subsequent treatment with
hydrazine proceeded with reduced racemization. Similar
results were obtained with the carboxymethyl derivative
2. However, with compound 2 a 6:1 excess of L-Ser over
D-Ser was observed when hydrazine treatment was
performed after hydrolysis, while hydrazine treatment
prior to hydrolysis provided a 3:1 excess of L- relative to
D-Ser. These results allowed assignment of the stereo-
chemistry of the R-position as (S), which indicated that
L-guanidino serine was present in 1. The reduced recov-
ery when acid hydrolysis occurred before treatment with
hydrazine is probably due to decomposition of the guani-
dino acid to a keto-acid.23
Acid hydrolysis of 1 and derivatization with FDAA
allowed the stereochemistry at the R position of isoleucine
to be determined, but there was insufficient resolution
of isoleucine and allo-isoleucine to allow unambiguous
determination of the stereochemistry at the â position.
(26) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(27) Ford, P. W.; Gustafson, K. R.; McKee, T. C.; Shigematsu, N.;
Maurizi, L. K.; Pannell, L. K.; Williams, D. E.; de Silva, E. D.; Lassota,
P.; Allen, T. M.; Van Soest, R.; Anderson, R. J .; Boyd, M. R. J . Am.
Chem. Soc. 1999, 121, 5899-5909.
(28) Thomas, D. W.; Das, B. C.; Gero, S. D.; Lederer, E. Biochem.
Biophys. Res. Commun. 1968, 32, 519-525.
J . Org. Chem, Vol. 69, No. 9, 2004 3039

Milanowski et al.
Gym n a n gia m id e (1). Amorphous white solid; [R]D
-32.5 (c 0.24, MeOH); UV (MeOH) λmax (log ꢀ) 258 (2.99) nm;
IR (film, NaCl) υmax 3333, 1655, 1542, 1455, 1204, 1139, 843,
tive 3 were approximately 7-fold less potent cytotoxins
relative to the native peptide 1. This indicated that the
amphoteric nature of 1 alone is not responsible for its
modest cytotoxic activity relative to that of the dolast-
atins. An additional feature of gymnangiamide (1) is that
both the dolaisoleuine and dolaproine residues that are
common among the dolastatins were found for the first
time in nature as their desmethyl deriviatives. This
difference in methylation pattern between 1 and the
dolastatins may account for some of its reduced cytotoxic
potency. In a synthetic study, Miyazaki et al.31 found that
the lack of methylation observed in the Ddil or Ddap
residues did not lead to significant loss of activity when
present individually, but they did not test analogues that
were missing methyl groups at both positions simulta-
neously.
Another point of interest raised by the similarity
between the dolastatins and 1 is the biosynthetic origin
of 1 in G. regae. Although originally isolated from the
marine mollusc Dolabella, the dolastatins are now be-
lieved to be of cyanobacterial origin, accumulating in
Dolabella as a result of dietary intake. In fact, a number
of dolastatins including dolastatin 10 have now been
isolated directly from cyanobacteria.19,29,32,33 The struc-
tural similarity between 1 and the dolastatins, suggests
that the ultimate origin of gymnangiamide (1) may be
from cyanobacteria in the diet or those living in associa-
tion with Gymnangium regae.
1
802, 723 cm-1; H NMR and 13C data, see Table 1; FABMS,
positive ion, m/z 750.5 [M + H]+, m/z 772.5 [M + Na]+;
HRFABMS, CsI-doped sample, m/z 882.3374 [M + Cs]+, calcd
for C36H59N7O10Cs 882.3377 (∆ -0.3 mmu). A positive result
for the presence of guanidine was achieved by spotting 1
(5-50 µg) onto TLC plates which were subsequently treated
with either FCNP or Sakaguchi spray reagents.21
Com p lete Acid Hyd r olysis. The peptide (50-200 µg per
hydrolysis) was dissolved in 6 N HCl (constant boiling, 0.5 mL),
degassed, and heated at 105-108 °C for 16 h under vacuum.
The solvent was removed under N2, and the residue washed
with H2O, frozen, and lyophilized to give the hydrolysis
product.
P r ep a r a tion a n d An a lysis of F DAA (Ma r fey’s) Der iva -
tives.26 Acid hydrolysate or appropriate standards were
transferred to an LC-MS vial and dried. The residue was
dissolved in 15 µL of 6% TEA (in MeCN-H2O, 1:1) and treated
with 7.5 µL of 1% N-(3-fluoro-4,6-dinitrophenyl)-L-alaninamide
(FDAA) in acetone at 40 °C for 60 min. The reaction mixture
was diluted with 45 µL of ddH2O and analyzed by LC-MS. A
20-µL aliquot was applied to a C8 column (Zorbax 300SB, 2.1
mm × 150 mm, 5 µm) eluting with 5% MeCO2H for 5 min
followed by a linear gradient (0-50%) of MeCN in 5% MeCO2H
over 25 min (0.25 mL/min, 50 °C). For analysis of derivatized
serine the elution was 15 min with 5% MeCO2H followed by
0-50% MeCN over 15 min. FDAA derivatized amino acids
were detected by absorption at 340 nm and by MSD (positive
ion, mass range 100-1000 Da).
P r ep a r a t ion a n d An a lysis of GITC Der iva t ives.27
Acid hydrolysate or appropriate standards were transferred
to an LC-MS vial and dried. The residue was taken up in
15 µL of 6% TEA (in MeCN-H2O, 1:1) and treated with
7.5 µL of 1% 2,3,4,6-tetra-O-acetyl-â-D-glucopyranosyl isothio-
cyanate (GITC) in acetone at room temperature for 10 min.
The reaction mixture was diluted with 45 µL of ddH2O and
analyzed by LC-MS as described for FDAA derivatives
above. GITC derivatized amino acids were detected by absorp-
tion at 254 nm and by MSD (positive ion, mass range 100-
1000 Da).
P r ep a r a tion of R-Gu a n id in o Ser in e Sta n d a r d s. A solu-
tion of 210 mg of D-serine (2 mmol) and 293 mg of 1H-pyrazole-
1-carboxamidine HCl (2 mmol) in 2 mL of 1.0 M Na2CO3 was
stirred for 16 h at room temperature. The reaction mixture
was then diluted with 6 mL of H2O and extracted with 3 × 4
mL of Et2O to yield R-guanidino D-serine in the aqueous layer.
Standards of R-guanidino L-serine were prepared in an analo-
gous manner starting with L-serine.
Ster eoch em ica l Deter m in a tion of Gu a n id in o Ser in e.
Gymnangiamide (1) or its carboxymethyl derivative (2; 50 µg)
was treated with 200 µL of 1:1 hydrazine hydrate-ddH2O for
60 min at 95 °C.28 The samples were cooled and dried by
passage of a stream of N2, and then washed with H2O (0.5
mL), frozen, and lyophilized. The hydrazine-treated samples
were then hydrolyzed with 6 N HCl as described above. In a
parallel set of reactions acid hydrolysis was performed prior
to treatment with hydrazine hydrate. The hydrolysis/hydrazi-
nolysis products (15 µg) and authentic standards of D- and
L-serine were derivatized with FDAA and analyzed by LC-MS.
The retention times of the standards were tR ) 13.81 min for
L-Ser and tR ) 15.36 min for D-Ser. The sample of 1 treated
with hydrazine followed by hydrolysis provided racemic Ser
[tR ) 13.48 and 15.07 min, ca. 1:1 ratio (area, EIC, m/z 358)].
The sample of 1 that was hydrolyzed prior to treatment with
hydrazine gave L-Ser (tR ) 13.50) in greater than 10-fold excess
over D-Ser but with significantly reduced overall yield. Com-
pound 2 provided a 3:1 excess of L- over D-Ser (tR ) 13.77 and
15.39) when hydrazine treatment was followed by acid hy-
drolysis and a 6:1 excess of L- over D-Ser when acid hydrolysis
preceded treatment with hydrazine (tR ) 13.78 and 15.32).
Exp er im en ta l Section
An im a l Ma ter ia l. Samples of the feathery marine hydroid
Gymnangium regae (order Conica, family Aglaopheniidae)
were collected in 1994 at a depth of -12 m off Luzon in the
Philippines. A voucher specimen for this collection (0CDN2622)
is maintained at the Smithsonian Institution, Washington,
D.C. The genus Gymnangium was recently synonymous with
Aglaophenia. Members of this genus are found in the Indo
Pacific, Caribbean, Mediterranean, and North Atlantic regions
and are fan or feather-like in appearance.
Extr a ction a n d Isola tion . The samples were frozen,
ground into a coarse powder (369.8 g, dry weight), and
extracted with H2O to yield 45.0 g of crude extract after
lyophilization. A 12.8-g portion of the crude aqueous extract
was dissolved in H2O, applied to a wide-pore C4 column (5 cm
× 8 cm), and sequentially eluted with H2O (100%), H2O-
MeOH (2:1), H2O-MeOH (1:2), and MeOH (100%) under
vacuum. The H2O-MeOH (1:2) fraction (127.1 mg) was puri-
fied by gel permeation chromatography on Sephadex LH-20
(3 cm × 100 cm) eluting with MeOH-H2O (7:3), followed by
C
18 HPLC (Dynamax, 1 cm × 25 cm, 8 µm) employing a linear
gradient from 0 to 100% MeCN in H2O (+0.1% TFA) over 30
min (3 mL/min) with detection at 220 nm yielding compound
1 (2.4 mg, tR 17.4 min). Reisolation from associated fractions
and extraction of the remaining crude material provided an
additional 4.8 mg of 1 (total yield 0.0068% based on dry
weight).
(29) Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald,
C. L.; Schmidt, J . M. J . Org. Chem. 1989, 54, 6005-6006.
(30) Pettit, G. R.; Singh, S. B.; Hogan, F.; Lloyd-Williams, P.; Herald,
D. L.; Burkett, D. D.; Clewlow, P. J . J . Am. Chem. Soc. 1989, 111,
5463-5465.
(31) Miyazaki, K.; Kobayashi, M.; Natsume, T.; Gondo, M.; Mikami,
T.; Sakakibara, K.; Tsukagoshi, S. Chem. Pharm. Bull. 1995, 43, 1706-
1718.
(32) Luesch, H.; Moore, R. E.; Paul, V. J .; Mooberry, S. L.; Corbett,
T. H. J . Nat. Prod. 2001, 64, 907-910.
(33) Horgen, F. D.; Kazmierski, E. B.; Westenburg, H. E.; Yoshida,
W. Y.; Scheuer, P. J . J . Nat. Prod. 2002, 65, 487-491.
3040 J . Org. Chem., Vol. 69, No. 9, 2004

Gymnangiamide
Liga n d Exch a n ge Ch ir a l HP LC of Isoleu cin e. The
hydrolysis product (50 µg) in ddH2O was applied to a ligand
exchange column (Phenomenex, Chirex 3126 (D)-Penicillamine,
4.6 mm × 250 mm) eluting with 2 mM Cu2+ sulfate (1.0 mL/
min) with detection at 254 nm. Isoleucine standards with
retention times (min) shown in parentheses are as follows:
L-allo-Ile (50.75), L-Ile (59.74), d-allo-Ile (85.81), and D-Ile
(102.27). The observed retention time in the hydrolysate of 1
was 59.84 min corresponding to L-Ile.
Meth yla tion a n d Absolu te Ster eoch em istr y of O-Des-
m eth yld ola p r oin e. An aliquot of 1 (100 µg) was dried in a
small vial to which 100 µL of 0.25 M NaH in DMSO was added.
The vial was purged with N2, capped, and allowed to sit 1.5 h.
MeI (5 µL) was then added, and the vial was again purged
with N2 and allowed to sit an additional 16 h in the dark. The
reaction was quenched by the addition of 175 µL of ddH2O and
neutralized with 25 µL of 1.2 N HCl. The reaction mixture
was dried under N2 and then hydrolyzed with 6 N HCl as
above. The hydrolysate (15 µg per reaction) was derivatized
separately with FDAA and GITC and analyzed by LC-MS. The
observed retention times for methylated Ddap in the hydroly-
sate were 27.86 min for FDAA and 24.68 min for GITC. An
authentic standard of Dap30 was similarly derivatized and
analyzed by LC-MS, yielding retention times of 27.80 min for
FDAA and 24.65 min for GITC.
Meth yla tion a n d Absolu te Ster eoch em istr y of N-Des-
m eth yld ola isoleu cin e. Methylation on the nitrogen of the
Ddil residue in the intact peptide was not successful. The free
amino acids obtained by acid hydrolysis (45 µg) were acetylated
by treatment with acetic anhydride (200 µL) in 800 µL of
MeOH and 40 µL of TEA for 60 min at room temperature. The
acetylated amino acids were dried under N2 in a small vial to
which 300 µL of 0.05 M NaH in DMSO was added. MeI (5 µL)
was added, the vial was purged with N2 and capped, and the
reaction mixture was stirred for 24 h at ambient temperature
away from light. The reaction was quenched and neutralized
with the addition of 300 µL of ddH2O and 25 µL of 1.2 M HCl.
The resulting solution was then dried under N2 and hydrolyzed
with 0.5 mL of 6 N HCl (constant boiling) for 60 min at 105-
108 °C to remove the acetate. The hydrolysate was dried under
N2, brought up in 0.5 mL of H2O, and lyophilized. The
hydrolysate was then derivatized with either FDAA or GITC
and analyzed by LC-MS. The observed retention times of
methylated Ddil were 26.49 min for the FDAA and 27.53 min
for the GITC derivative. An authentic standard of Dil30 was
similarly derivatized and analyzed by LC-MS, yielding reten-
tion times of 26.56 min with FDAA and 27.61 min with GITC.
Absolu te Ster eoch em istr y of P h en ylser in e. (a ) P r ep a -
r a tion of P h en ylser in e Sta n d a r d s. dl-threo-Phenylserine
(76 mg) in 1 mL of 9% Na2CO3 and 90 mg of FMOC-Cl in 1
mL of dioxane were chilled on ice and combined. The mixture
was allowed to warm to room temperature with stirring, which
was continued for 60 min. The reaction mixture was then
diluted with 8 mL of ddH2O and extracted with ether (3 × 4
mL), discarding the organic layers. The aqueous layer was
acidified (pH 2) with concentrated HCl to afford a white
precipitate. The suspension was extracted with EtOAc (4 × 4
mL) and the organic layers combined and dried under N2 to
provide a mixture of FMOC-protected D- and L-threo-phe-
nylserines.31 The FMOC derivatives were separated by chiral
HPLC on a Sumipax OA-3100 DNPAC-(S)-V column (Regis,
4.6 mm × 150 mm, 5 µm). Elution was isocratic, using 30 mM
ammonium acetate in MeOH at 1 mL/min with detection at
265 nm. Chiral HPLC resulted in the recovery of two compo-
nents, peak A (tR ) 6.91 min) and peak B (tR ) 7.26 min); peak
B was subjected to a second round of purification. The
individual derivatives were dried under N2, 0.5 mL of ddH2O
was added, and the samples were lyophilized. The resulting
powders were deprotected with 1 mL of 20% piperidine in DMF
for 3 h at ambient temperature. The deprotecting reagents
were removed under N2 and the residue taken up in 1 mL of
ddH2O and extracted with ether (3 × 1 mL), discarding the
organic layers. The aqueous layer was acidified with 3 drops
of concentrated HCl (pH <2) and extracted with EtOAc
(3 × 1 mL), discarding the organic layers. The aqueous layers
were dried under N2, brought up in 0.5 mL of ddH2O, and
lyophilzed to provide D- and l-threo-phenylserine HCl. Peak A
provided 1.4 mg of a relatively pure isomer (ca. 95% based
on analysis of FDAA-derivatized sample) with [R]D +22.1 (c
0.14, H2O), which is in agreement for d-threo-phenylserine.35,36
Peak B provided 3.4 mg of the isomer with [R]D -20.3 (c 0.34,
H2O), which is in agreement for L-threo-phenylserine.37 The
erythro isomers were obtained by acid-catalyzed racemization
of the commercial DL-threo-phenylserine. DL-threo-Phenylserine
(200 µg) was heated 48 h in 6 N HCl (constant boiling) at 125-
130 °C, and then dried under N2 and lyophilized prior to
analysis.
(b) Deter m in a tion of P h en ylser in e Absolu te Ster eo-
ch em istr y in 1. Acid hydrolysate of 1 (20 µg) and phenylserine
standards were derivatized with FDAA and analyzed by LC-
MS. Retention times (min) of FDAA-derivatized standards are
the following: L-threo-phenylserine (21.04), d-threo-phe-
nylserine (24.16), and erythro-phenylserine isomers (19.99,
23.14). The observed retention time for phenylserine in the
hydrolysate of 1 was 21.03 min, corresponding to l-threo-
phenylserine.
P r ep a r a tion of Meth yla ted Der iva tive 2.25 Gymnangia-
mide (0.5 mg) was brought up in 1.0 mL of benzene and 250
µL of MeOH and treated with 150 µL of (trimethylsilyl)-
diazomethane (TMSCHN2). The reaction was stirred for 18 h
at room temperature away from light. The mixture was dried
under N2, washed with 0.5 mL of MeOH, and again dried
under N2. The reaction mixture was analyzed by LC-MS: C8
column (Zorbax 300SB, 2.1 mm × 150 mm, 5 µm) eluting with
a linear gradient (0-100%) of MeCN in 5% MeCO2H over 60
min (0.20 mL/min, 40 °C). The carboxymethyl addition product
(2) was observed (m/z 764.7 [M + H]+, tR ) 21.33).
P r ep a r a tion of th e Dim eth ylp yr im d in e Der iva tive 3.22
Gymnangiamide (0.5 mg) was methylated as above and then
further derivatized by treatment with 500 µL of 2,4-pentanedi-
one containing 12 mg/mL of NaHCO3 for 30 min at 130 °C to
give the dimethylpyrimidinyl derivative 3. The product was
chromatographed by LC-MS as above (m/z 828.5 [M + H]+, tR
) 25.17).
Cytotoxicity Assa y. Chromatography fractions and puri-
fied 1 were made up in DMSO-H2O (1:1) and assayed in an
in vitro cytotoxicity assay against COLO-205 and OVCAR-3
cell lines to direct purification. Differential cytotoxicity was
evaluated utilizing a panel of 10 human tumor cell lines.
Following each cell line is the tumor type and IC50 observed
for 1: COLO-205 (colon, 4.7 µg/mL), H460 (lung, 0.46 µg/mL),
K562 (leukemia, 11.5 µg/mL), MOLT-4 (leukemia, 5.8 µg/mL),
A549 (lung, 5.8 µg/mL), and MALME-3M (melanoma, 9.6 µg/
mL). With the LOX (melanoma), OVCAR-3 (ovarian), and
SNB-19 (cns) cell lines, growth inhibition was observed but
an IC50 could not be determined, while no inhibition was
observed with MCF-7 (breast) at a high test concentration of
15 µg/mL. Relative cytotoxicity for compounds 1-3 was
determined by using the IC-2WT murine cell line,38 which
provided IC50 values for 1 (1.7 µg/mL), 2 (11.2 µg/mL), and 3
(12.5 µg/mL). Experimental details of the 2-day antiprolifera-
tion assay employed are published elsewhere.39
(34) Carpino, L. A.; Han, G. Y. J . Org. Chem. 1972, 37, 3404-3409.
(35) Herbert, R. B.; Wilkinson, B.; Ellames, G. J . Can. J . Chem.
1994, 72, 114-117.
(36) Koskinen, A. M. P.; Hassila, H.; Myllymaki, V. T.; Rissanen,
K. Tetrahedron Lett. 1995, 36, 5619-5622.
(37) Vogler, K. Helv. Chim. Acta 1950, 33, 2111-2117.
(38) Hashimoto, K.; Tsujimura, T.; Moriyama, Y.; Yamatodani, A.;
Kimura, M.; Tohya, K.; Morimoto, M.; Kitayama, H.; Kanakura, Y.;
Kitamura, Y. Am. J . Pathol. 1996, 148, 189-200.
(39) Bokesch, H. R.; Blunt, J . W.; Westergaard, C. K.; Cardellina,
J . H., II; J ohnson, T. R.; Michael, J . A.; McKee, T. C.; Hollingshead,
M. G.; Boyd, M. R. J . Nat. Prod. 1999, 62, 633-635.
J . Org. Chem, Vol. 69, No. 9, 2004 3041

Milanowski et al.
Ack n ow led gm en t. We thank D. Newman for coor-
dinating collections, P. L. Colin (CRRF) for sample
collections, T. McCloud for sample extractions, and T.
J ohnson, J . Wilson, and N. Oku for cytotoxicity evalu-
ations. We also thank G. Robert Pettit and his col-
leagues at Arizona State University for kindly providing
authentic Dap and Dil standards. This project was
supported by NCI contract No. N01-C0-12400. The
content of this publication does not necessarily reflect
the views or policies of the department of Health and
Human Services, nor does mention of trade names,
commercial products, or organization imply endorse-
ment by the U.S. government.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal details and NMR spectra for gymnangiamide. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O0303113
3042 J . Org. Chem., Vol. 69, No. 9, 2004