Polytheonamides A and B, highly cytotoxic, linear polypeptides with unprecedented structural features, from the marine sponge, Theonella swinhoei

Article abstract of DOI:10.1021/ja045749e

Polytheonamides A and B are highly cytotoxic polypeptides with 48 amino acid residues isolated from the marine sponge, Theonella swinhoei. The structure of polytheonamide B was determined by spectral and chemical methods, especially extensive 2D NMR experiments, which resulted in the unprecedented polypeptide structure; the N-terminal glycine blocked with a 5,5-dimethyl-2-oxo-hexanoyl group, the presence of eight tert-leucine, three β-hydroxyvaline, six γ-N-methylasparagine, two γ-N-methyl-β-hydroxyasparagine, and β,β-dimethymethionine sulfoxide residues. More significantly, it has the sequence of alternating D-and L-amino acids. Polytheonamide A is an epimer of polytheonamide B differing only in the stereochemistry of the sulfoxide of the 44^th residue.

Full text of DOI:10.1021/ja045749e

Published on Web 12/13/2004
Polytheonamides A and B, Highly Cytotoxic, Linear
Polypeptides with Unprecedented Structural Features, from
the Marine Sponge, Theonella swinhoei
Toshiyuki Hamada,† Shigeki Matsunaga, Gen Yano, and Nobuhiro Fusetani*
Contribution from the Laboratory of Aquatic Natural Products Chemistry, Graduate School of
Agricultural and Life Sciences, The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Received July 15, 2004; E-mail: anobu@mail.ecc.u-tokyo.ac.jp
Abstract: Polytheonamides A and B are highly cytotoxic polypeptides with 48 amino acid residues isolated
from the marine sponge, Theonella swinhoei. The structure of polytheonamide B was determined by spectral
and chemical methods, especially extensive 2D NMR experiments, which resulted in the unprecedented
polypeptide structure; the N-terminal glycine blocked with a 5,5-dimethyl-2-oxo-hexanoyl group, the presence
of eight tert-leucine, three â-hydroxyvaline, six γ-N-methylasparagine, two γ-N-methyl-â-hydroxyasparagine,
and â,â-dimethymethionine sulfoxide residues. More significantly, it has the sequence of alternating D-
and L-amino acids. Polytheonamide A is an epimer of polytheonamide B differing only in the stereochemistry
of the sulfoxide of the 44th residue.
Introduction
mides A and B including the sequence specific stereochemical
assignments of amino acid residues are described.
The marine sponge Theonella swinhoei, with a yellow interior
and collected from Hachijo-jima Island, 300 km south of Tokyo,
is extraordinarily rich in bioactive metabolites of unusual
structures and potent activities, including serine protease inhibi-
tory cyclotheonamides,2 nazumamide A,3 and pseudotheona-
mides,4 cytotoxic onnamides,5 theopederins,6 and orbiculamide
A,7 and antifungal aurantosides.8 In 1994, we reported the
isolation and structure determination of the highly cytotoxic
polypeptides, polytheonamides, from the same sponge.9-11
During the stereochemical study of polytheonamide B, we
realized that the previously proposed structures were incorrect.
In this report, the revised complete structures of polytheona-
Results
The sponge extract was subjected to solvent partitioning, gel
filtration, and reversed-phase HPLC to afford polytheonamides
A, B, and C (3.8, 5.2, and 1.0 × 10-4 % wet weight,
respectively). Polytheonamides A, B, and C were cytotoxic
against P388 murine leukemia cells with IC50 values of 78, 68,
and 68 pg/mL, respectively.
The structure elucidation was commenced with polytheona-
mide B, the major polytheonamide. The electrospray ionization
mass spectrum (ESIMS) exhibited ions at m/z 1700.9 (M +
3Na)3+ and 2539.8 (M + 2Na)2+, in agreement with the
molecular weight of 5032, which was supported by the (M +
H)+ ion at m/z 5033 in the FABMS. The 1H NMR spectrum of
polytheonamide B (Figure 1A) exhibited numbers of signals in
the R-methine and amide regions, whereas the 13C NMR
spectrum (Figure 1B) gave a cluster of amide signals, indicating
its polypeptidic nature.
Component Amino Acids. Amino acid analysis of poly-
theonamide B showed the presence of Asp (7.9 residues), Thr
(3.9), Ser (1.5), Glu (2.0), Gly (10.0), Ala (8.9), Val (4.3), and
Ile (2.3).12 In addition to these common amino acids, there were
several unidentified peaks in the chromatogram: an acidic amino
acid eluted faster than Asp; a neutral amino acid with a retention
time close to that of Cys; and a neutral amino acid with a slightly
longer retention time than that of Ile. The peaks corresponding
to Thr, Ser, and Glu were broad, suggesting that they were
overlapped with peaks of unassigned amino acids.
† Present address: RIKEN Genomic Sciences Center, 1-7-22 Suehiro-
cho, Tsurumi-ku, Yokohama 230-0045, Japan, and Division of Protein Folds
Research, Graduate School of Integrated Science, Yokohama City Univer-
sity, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
(1) Part 129 of the Bioactive Marine Metabolites series. Part 128: Matsunaga,
S.; Miyata, Y.; van Soest, R. W. M.; Fusetani, N. J. Nat. Prod. 2004, 67,
1758-1760.
(2) (a) Fusetani, N.; Matsunaga, S.; Matsumoto, H.; Takebayashi, Y. J. Am.
Chem. Soc. 1990, 112, 7053-7054. (b) Nakao, Y.; Matsunaga, S.; Fusetani,
N. Bioorgan. Med. Chem. 1995, 3, 1115-1122.
(3) Fusetani, N.; Nakao, Y.; Matsunaga, S. Tetrahedron Lett. 1991, 32, 7073-
7074.
(4) Nakao, Y.; Matsuda, A.; Matsunaga, S.; Fusetani, N. J. Am. Chem. Soc.
1999, 121, 2425-2431.
(5) Matsunaga, S.; Fusetani, N.; Nakao, Y. Tetrahedron 1992, 48, 8369-8376.
(6) (a) Fusetani, N.; Sugawara, T.; Matsunaga, S. J. Org. Chem. 1992, 57,
3828-3832. (b) Tsukamoto, S.; Matsunaga, S.; Fusetani, N.; Toh-e A.
Tetrahedron 1999, 55, 13697-13702.
(7) Fusetani, N.; Sugawara, T.; Matsunaga, S.; Hirota, H. J. Am. Chem. Soc.
1991, 113, 7811-7812.
(8) Matsunaga, S.; Fusetani, N.; Kato, Y.; Hirota, H. J. Am. Chem. Soc. 1991,
113, 9690-9692.
(9) Hamada, T.; Sugawara, T.; Matsunaga, S.; Fusetani, N. In Sponges in Time
and Space; van Soest, R. W. M., van Kempen, T. M. G., Braekman, J.-C.,
Eds.; A. A. Balkema: Rotterdam, 1994, pp 453-457.
(10) Hamada, T.; Sugawara, T.; Matsunaga, S.; Fusetani, N. Tetrahedron Lett.
1994, 35, 719-720.
(12) Thr and aThr exhibited an identical retention time in the amino acid analysis.
The molar equivalents are not precise due to peak overlapping with unusual
amino acids. For the chromatogram of amino acid analysis, see Figure S14.
(11) Hamada, T.; Sugawara, T.; Matsunaga, S.; Fusetani, N. Tetrahedron Lett.
1994, 35, 609-612.
9
110
J. AM. CHEM. SOC. 2005, 127, 110-118
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Polytheonamides, Highly Cytotoxic Polypeptides
A R T I C L E S
a pair of mutually coupled methylenes and a ketone resonance.
On reduction with NaBH4, polytheonamide B afforded a 2:1
mixture of dihydropolytheonamides B1 and B2, both of which
exhibited an oxymethine signal. The COSY spectrum of
dihydropolytheonamide B1 displayed signals assignable to a
partial structure -CδH2-CγH2-CâH(OH)- [δH 3.80 (Hâ), 1.60,
1.40 (Hγ,γ′), 1.29, 1.17 (Hδ,δ′); δC 71.5 (Câ), 29.6 (Cγ), 39.0
(Cδ)]. Although the methyl region of the HMBC spectrum of
polytheonamide B was highly crowded, cross-peaks were
observed from a singlet methyl (δH 0.87) to a methylene carbon
at δ 36.6 (Cδ) and to a nonprotonated carbon at δ 29.6, thus
suggesting the linkage of Cδ to a tert-butyl group. HMBC cross-
peaks were also observed between the amide and methylene
protons of the N-terminal Gly residue and the CR carbon (δ
161.2) of the blocking group. Although no NOESY cross-peaks
between Gly1 and the blocking group were detected, probably
due to an unfavorable correlation time for this portion of the
molecule, the ROESY cross-peak between the amide proton
(Gly1) and the oxygenated methine proton in dihydropoly-
theonamide B1 was observed, thereby disclosing that the
N-terminal Gly of polytheonamide B was blocked with a 5,5-
dimethyl-2-oxo-hexanoyl group.
Figure 1. 1H (A) and 13C NMR spectra (B) of polytheonamide B in
DMSO-d6.
To identify the uncommon amino acids, the acid hydrolysate
was directly analyzed by 2D NMR; the hydrolysate exhibited
the much simpler NMR spectra than those of the intact peptide,
which was due to the convergence of signals. In addition to the
common amino acids mentioned above, aThr,13 threo-â-
hydroxyaspartic acid (OHAsp),14 â-methylglutamic acid (âMe-
Glu),15 t-Leu,16 â-hydroxyvaline (OHVal),17 and â-methyliso-
leucine (âMeIle)18 could be assigned. Since âMeIle was not
thoroughly assigned due to the heavily crowded aliphatic signals,
this amino acid was isolated and identified from the hydroly-
sate.19 Intense signals assignable to methylamine were also
observed.20
Structures of the N-Terminus and 44th Amino Acid
Residues. Previously, the N-terminal-blocking group and the
44th residue were assigned as carbamoyl and γ-hydroxy-t-
leucine, respectively, on the basis of NMR data.10,11 In addition,
polytheonamide B was assigned as a tripropylammonium salt
in order to account for the MS data and due to the NMR signals
for tripropylamine. Subsequently, tripropylamine was found to
be a contaminant from the n-PrOH used for HPLC. In the
meantime, we realized the presence of a functional group
reducible with NaBH4 which prompted the reinvestigation of
the total structure. Inspection of the 2D NMR spectra indicated
Since the N-terminal structure was revised and tripropylamine
was not contained in the molecule, the 44th residue, which had
previously been assigned as γ-OH-t-Leu, had to be revised.
Interpretation of the HMBC data showed a cross-peak between
a singlet methyl (δ 2.52) and the methylene carbon at δ 63.3,
which was originally assigned to an oxygenated methylene.
However, these chemical shift values were more consistent with
two carbons which were separated by a sulfoxide group, which
was also in accordance with MS data. In fact, the elemental
analysis of polytheonamide B demonstrated the presence of one
sulfur atom. Therefore, the 44th residue was â,â-dimethylme-
thionine sulfoxide [Me2Met(O)]. This amino acid and 5,5-
dimethyl-2-oxo-hexanoic acid were not detected in the acid
hydrolysate of polytheonamide B.
Sequencing of Amino Acids. To sequence the amino acids
in polytheonamide B using NMR spectroscopy solvents were
required which gave sharp and well-dispersed 1H NMR signals,
and high-quality NOESY spectra; CDCl3/CD3OH (1:1) and
DMSO-d6 satisfied these criteria. In CDCl3/CD3OH (1:1), the
NOESY cross-peaks were observed between adjacent residues
and between sequentially distant residues. However, this feature
also complicated the sequence analysis. By contrast, poly-
theonamide B appeared to adopt a random-coil conformation
in DMSO-d6, in which NOESY cross-peaks were observed only
between adjacent residues, thereby facilitating sequential signal
assignments. The number of amino acid residues was established
by interpretation of the HOHAHA spectrum as follows: OHAsx
(2), Asx (8), Thr/aThr (1 each), Ser (1), Glx (1), âMeGlx (1),
Ala (7), Val (3), and Ile (2). The residues that contained a
quaternary carbon in the side chain were analyzed on the basis
of intra-residual NOESY cross-peaks which were more intense
than inter-residual cross-peaks, allowing the determination of
eight t-Leu, one âMeIle, three OHVal, and one Me2Met(O) unit
(Figure 2). Sequence analysis of the peptide chain in DMSO-
d6 was unexceptional once the types and numbers of components
were established (Figure 3, Table 1).
(13) In addition to an A3MX system for Thr (δ 1.21, 3.82, and 4.28), there was
another A3MX system with similar chemical shift values (δ 1.15, 3.94,
and, 4.25), which could be assigned as aThr.
(14) An AB system at δ 4.75 and 4.28 indicated the presence of threo-â-
hydroxyAsp. [(a) Kato, T.; Hinoo, H.; Terui, Y.; Kikuchi, J.; Shoji, J. J.
Antibiot. 1988, 41, 719-725. (b) Tymiak, A. A.; McCormick, T. J.; Unger.
S. E. J. Org. Chem. 1989, 54, 1149-1157. (c) ElDin, A. L. M. S.; Kyslik,
P.; Stephan, D.; Abdallah, M. A. Tetrahedron, 1997, 53, 12539-12552.]
(15) A methine proton at δ 2.50 exhibited COSY cross-peaks with an R-methine
signal at δ 3.98, a methylene at δ 2.39 and 2.53, and a methyl at δ 0.95.
(Debono, M.; Barnhart, M.; Carrell, C. B.; Hoffmann, J. A.; Occolowitz,
J. L.; Abbott, B. J.; Fukuda, D. S. J. Antibiot. 1987, 40, 761-777.)
(16) There was a large methyl singlet at δ 0.96, which exhibited HMBC
correlations with carbons at δ 26.0, 33.0, and 62.5. The R-methine proton
attached to a carbon at δ 62.5 resonated at δ 3.32, as a singlet.
(17) The HMBC spectra revealed that both singlet methyls at δ 1.17 and 1.32
were correlated with carbons at δ 62.9 and 70.6. (Ikai, K.; Takesako, K.;
Shiomi, K.; Moriguchi, M.; Umeda, Y.; Yamamoto, J.; Kato, I. J. Antibiot.
1991, 44, 925-933.)
(18) (a) Gulavita, N. K.; Gunasekera, S. P.; Pomponi, S. A.; Robinson, E. V. J.
Org. Chem. 1992, 57, 1767-1772. (b) Li, H.; Matsunaga, S.; Fusetani, N.
J. Med. Chem. 1995, 38, 338-343. (c) Rashid, M. A.; Gustafson, K. R.;
Cartner, A. K.; Shigematsu, N.; Pannell, L. K.; Boyd, M. R. J. Nat. Prod.
2001, 64, 117-121.
(19) 1H NMR data for the isolated âMeIle: δ 0.78 (3H, t), 0.88 (3H, s), 0.89
(3H, s), 1.32 (2H, q), and 3.40 (1H, s).
There were eight doublet N-methyl resonances attributable
to N-methylation in the side-chain amide groups. All of the
(20) 1H NMR data for the isolated MeNH2: δ 2.44 (3H, s).
9
J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005 111

A R T I C L E S
Hamada et al.
Figure 2. Amide-aliphatic proton (A and B) and amide-R-proton (C) regions of the NOESY spectrum of polytheonamide B in DMSO-d6 at 300K. Assigned
cross-peaks are indicated in the spectrum.
carbonyl groups in the side chain were linked to either amide
or N-methylamide moieties. Sequence specific assignment of
these groups was accomplished by the interpretation of NOESY
data, which disclosed that residues 15, 21, 27, 29, 33, 35, 37,
and 39 were N-methylated in the side chain.
Stereochemistry of Amino Acids. The stereochemistry of
the amino acid residues in polytheonamide B was determined
by GC analysis on a chiral stationary phase22 and HPLC analysis
of FDAA derivatives (Marfey analysis).23 Thr, Ile, Glu, Val,
and âMeIle were found to be in the L-form, while OHAsp, Ser,
and aThr were in the D-form. A 2S,3S-stereochemistry was
assigned for âMeGlu by GC analysis.24 The remaining amino
acids were mixtures of both forms: Ala (D/L ) 2:5), t-Leu (4:
4), Asp (7:1), and OHVal (2:1). Consequently, it was necessary
to assign the stereochemistry of these residues in a sequence-
specific manner in order to complete the structure elucidation
of polytheonamide B. To solve this issue, attempts were made
to digest polytheonamide B with a variety of proteases; this
was not successful. In contrast, mild acid hydrolysis worked
(21) Wu¨thrich, K. NMR of Proteins & Nucleic Acids; Wiley, New York, 1986.
(22) Frank, H.; Nicholson, G. J.; Bayer, E. J. Chromatogr. Sci. 1977, 15, 174-
(23) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(24) Mauger, A. B. J. Org. Chem. 1981, 46, 1032-1035.
176.
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Polytheonamides, Highly Cytotoxic Polypeptides
A R T I C L E S
Figure 3. Amide-amide proton region of the NOESY spectrum of polytheonamide B in DMSO-d6 at 300K. Sequential NOEs are indicated in the spectrum.
well; polytheonamide B was treated with 4N HCl in EtOH at
70 °C for 30 min to afford a mixture of fragment peptides which
was separated by reversed-phase HPLC to afford 120 fractions.
Each fraction was analyzed by tandem-FABMS. In this way,
the purities of the fractions could be checked and at the same
time the amino acid sequence of the fragment peptides
obtained.25 The results of the analysis are summarized in Table
2.
Eleven fractions were chosen for stereochemical study of the
component amino acids. All of these fractions were directly
subjected to stereochemical study, except for fraction 46 which
was dansylated and separated into fractions 46A and 46B. The
chiralities of amino acid residues were determined as follows:
(1) The stereochemistry of the amino acids in the acid
hydrolysate of the dansylated derivative was analyzed by either
Marfey’s method or by chiral GCMS analysis; (2) the chirality
of the N-terminal residue of a dansyl derivative was determined
by HPLC on a chiral stationary phase; (3) by one cycle of
Edman degradation, the N-terminal residue of a peptide was
removed and the second residue from the N-terminus was
analyzed as a dansyl derivative.26
were derived from the Marfey analysis of dansylated fractions
46A, 46B, and 36.
Although fragments containing any of eight residues from
the C-terminus could not be obtained, fortunately des-Thr48-
dihydropolytheonamide B2 could be isolated during the puri-
fication of dihydropolytheonamide B2.27 GC analysis of its acid
hydrolysate indicated the absence of L-Thr, thereby accom-
modating L-Thr at the C-terminus. The chirality of the amino
acids thus far obtained showed a sequence of alternating D and
L amino acids. If this rule is applicable to the whole peptide
sequence, then both Asn43 and Asn45 should be in the D-form,
although the amino acid analysis of polytheonamide B indicated
one of the two Asn residues to be L. The possibility of
racemization in the Asx residues during acidic hydrolysis was
explored.28 To address this issue, the hydrolysate obtained from
the hydrolysis of polytheonamide B with 6N DCl in D2O was
analyzed by chiral GCMS. As a result, the L-Asp fraction
exhibited ion peaks higher than those of D-Asp by 1 amu,
indicating that L-Asp was the product of inversion by acid.29
Thus, all of the Asx residues, including residues 43 and 45, in
polytheonamide B were originally D.
t-Leu4 was determined to be L by the N-terminal analysis of
dansylated fraction 100, whereas t-Leu5 was D from the
N-terminal analysis of fraction 90 after dansylation followed
by one cycle of Edman degradation. Marfey analysis of the
dansylated fraction 72 led to L-t-Leu6 and D-Ala7, while the
N-terminal analysis of dansylated fraction 65 disclosed D-t-Leu8.
L-t-Leu9 and D-Ala10 were assinged by the Marfey analysis of
dansylated fraction 52. Similarly, Marfey analysis of dansylated
fractions 23 and 39 resulted in L-Ala12, D-t-Leu13, L-Ala14,
D-Asp15, and L-Ala18, whereas GCMS analysis of dansylated
fraction 39 identified L-OHVal16. Thus, both OHVals at
positions 23 and 31 were D. L-t-Leu20, D-âMeGlu21, L-Ala24,
D-Asp27, L-t-Leu30, D-Asp33, D-Asp35, L-Ala38, and D-Asp39
Since â,â-dimethylmethionine sulfoxide was decomposed
during acid hydrolysis, its conversion to an acid-resistant
derivative was required in order to determine the stereochem-
istry. Advantage was taken of the structural resemblance
between this residue and methionine. In Met-containing peptides,
the C-terminal amide bond of Met is cleaved with CNBr, with
concomitant conversion to homoserine lactone.30 Therefore, it
was anticipated that the â,â-dimethylmethionine sulfoxide unit
in polytheonamide B could be converted to â,â-dimethyl-
(27) The C-terminal peptide bond was cleaved during evaporation of HPLC
solvent containing 0.05% TFA.
(28) (a) Brennan, T. V.; Clarke, S. Int. J. Pept. Protein Res. 1995, 45, 547-
553. (b) Goodlett, D. R.; Abuaf, P. A.; Savage, P. A.; Kowalski, K. A.;
Mukherjee, T. K.; Tolan, J. W.; Corkum, N.; Goldstein, G.; Crowther, J.
B. J. Chromatogr. A 1995, 707, 233-244.
(25) McLafferty, F. W. Science 1981, 214, 280-287.
(29) Because incorporation of deuterium was observed for both R- and â-
positions, EI-MS peaks were observed as a cluster of isotope ions. The
pattern of peaks for L-Asp shifted to the higher mass region by one amu.
(30) Gross, E. Methods Enzymol. 1967, 11, 238-255.
(26) (a) Tarr, G. E. Anal. Biochem. 1975, 63, 361-370. (b) Tarr, G. E. In
Methods of Protein Microcharacterization; Shively, J. E., Ed.; Humana
Press: Totawa, NJ; 1986, pp 155-194.
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J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005 113

A R T I C L E S
Hamada et al.
Table 1. 1H NMR Chemical Shift of Polytheonamide B in
the odd number residues are in the D form, whereas the even
number amino acids in the L form.
DMSO-d6 at 313Ka
residue
NH
CRH
CâH
others
The gross structure of polytheonamide A was assigned to be
identical with that of polytheonamide B by interpretation of the
2D NMR data in DMSO-d6 (Table 3). The 1H NMR spectra of
the two peptides were almost superimposable, except for minor
chemical shift differences observed in the signals for residues
41-48, among which the most notable differences were found
in the residue 44. The Marfey and chiral GC analyses of
polytheonamides A and B provided identical results. Therefore,
there were two possibilities: (1) polytheonamides A and B are
positional isomers in which stereochemistries of at least one
pair of amino acid residues were exchanged; (2) polytheona-
mides A and B are diastereomeric at the sulfoxide in residue
44. An accidental finding solved this problem. When the
deoxygenation products of polytheonamides A and B were
dissolved in n-PrOH/H2O (1:1) and the mixture left to stand at
room temperature for a week, they underwent air oxidation to
furnish a pair of HPLC peaks corresponding to polytheonamides
A and B in a 1:1 ratio. Furthermore, on oxidation with Oxone33
polytheonamides A and B afforded sulfones which were
Gly1
âMeIle2
8.50 3.83
7.76 4.33
CγH30.85; CγH30.89;
CγH21.30; CδH30.78
Gly3
8.25 3.63, 3.82
7.49 4.58
8.00 4.20
7.31 4.24
7.91 4.49
7.60 4.42
7.77 4.19
8.07 4.32
8.04 3.70, 3.80
7.88 4.46
7.68 4.24
8.13 4.31
8.08 4.65
7.67 4.22
8.06 3.78
7.96 4.31
8.18 3.66, 3.92
7.60 4.16
8.27 4.63
7.72 4.32
t-Leu4
t-Leu5
t-Leu6
Ala7
t-Leu8
t-Leu9
Ala10
Gly11
Ala12
t-Leu13
Ala14
Asm15
OHVal16
Gly17
Ala18
Gly19
t-Leu20
Asm21
âMeGln22
CγH30.90
CγH30.94
CγH30.86
1.21
CγH30.90
CγH30.93
1.23
1.23
CγH30.90
1.20
2.42, 2.55
1.09, 1.13
1.23
CγH30.88
2.41, 2.53
2.33
CγH30.82; CγH21.85,
2.17; NH2 6.73, 7.25
CγH31.11; CγH31.14
1
indistinguishable by HPLC, FABMS, and H NMR analysis.
Therefore, it was concluded that the two peptides are isomeric
at the sulfoxide moiety.
OHVal23
Ala24
Gly25
Gly26
Asm27
Ile28
7.97 4.28
7.87 4.35
8.16 3.78
7.99 3.78
7.99 4.68
7.67 4.30
1.24
2.43, 2.50
1.72
Discussion
CγH31.02; CγH21.35;
CδH30.75
In this paper, it was shown that polytheonamides A and B,
two highly cytotoxic constituents of the marine sponge T.
swinhoei, are polypeptides with unprecedented structural fea-
tures. The structure of the N-terminal blocking group is unusual
among natural products. Although the 5,5-dimethyl-2-oxo-
heptanoyl group was reported in synthetic compounds,34 this is
the first report of this component in a natural product. t-Leu is
a rare amino acid among nonribosomal peptides of microbial
or marine origin; this amino acid has so far been reported as a
component in bottromycins,35 discodermin A36 and related
peptides,18,37 and hemiasterlins.38 The presence of eight t-Leu
residues in one peptide is unprecedented. Other nonproteino-
genic amino acid residues in polytheonamides, such as âMeIle,
OHVal, âMeGlx, and Me2Met(O), are also methylated variants
of proteinogenic amino acids, as is t-Leu, a â-methylated variant
of valine. Me2Met(O) is a new amino acid. Another novel
feature of the component amino acids is the presence of eight
residues of γ-N-methylasparagine. This amino acid is a com-
ponent of the phycobiliproteins of cyanobacteria and red algae
and is reported to play a functional role in photosynthesis.39
Although reported previously as products of chemical transfor-
OHAsm29
t-Leu30
OHVal31
Gly32
Asm33
Ile34
7.93 4.68
7.27 4.46
7.95 4.31
8.07 3.71, 3.85
8.03 4.65
7.60 4.16
4.41
CγH30.88
CγH31.16; CγH31.16
2.42, 2.52
1.73
CγH31.05; CγH21.37;
CδH30.78
Asm35
Val36
OHAsm37
Ala38
Asm39
Val40
Ser41
8.14 4.68
7.62 4.13
7.95 4.65
7.69 4.25
7.99 4.65
7.66 4.22
8.01 4.40
7.68 4.23
8.25 4.66
2.41, 2.55
1.94
4.43
1.23
2.43, 2.58
2.03
CγH30.78; CγH30.78
CγH30.83; CγH30.83
CγH30.83; CγH30.83
3.58, 3.62
1.99
Val42
Asn43
2.42, 2.55 NH2 6.88, 7.29
CγH30.99; CγH31.07;
CγH22.70, 2.98; CꢀH32.52
2.47, 2.60 NH2 6.90, 7.36
Me2Met(O)44 7.61 4.60
Asn45
Gln46
8.47 4.61
7.93 4.29
2.12
CγH21.79, 1.93;
NH2 6.69, 7.16
CγH31.09
aThr47
Thr48
7.96 4.31
7.63 4.18
3.89
4.07
CγH31.05
a N-methyl amides in the side chain: NH/CH3, 7.62/2.58, 7.63/2.55, 7.65/
2.54, 7.66/2.52, 7.71/2.52, 7.72/2.52, 7.73/2.58, 7.79/2.58.
homoserine lactone, if this residue was reduced to a sulfide.
â,â-Dimethylhomoserine lactone was shown to be stable to acid
using a synthetic sample.31 Polytheonamide B was treated with
Me2S in the presence of NH4I32 to afford a deoxygenated
derivative which was then subjected to reaction with CNBr to
afford the expected truncated derivative (Scheme 1). GC analysis
of the product showed the presence of L-â,â-dimethyl-
homoserine lactone, thus completing the sequence-specific,
stereochemical assignments of polytheonamide B. Therefore,
(33) Trost, B. M.; Curran, D. P. Tetrahedron Lett. 1981, 22, 1287-1290.
(34) Krix, G.; Bommarius, A. S.; Drauz, K.; Kottenhahn, M.; Schwarm, M.;
Kula, M. -R. J. Biotechnol. 1997, 53, 29-39.
(35) Schipper, D. J. Antibiot. 1983, 36, 1076-1077.
(36) (a) Matsunaga, S.; Fusetani, N.; Konosu, S. J. Nat. Prod. 1985, 48, 236-
241. (b) Matsunaga, S.; Fusetani, N.; Konosu, S. Tetrahedron Lett. 1984,
25, 5165-5168. (c) Matsunaga, S.; Fusetani, N.; Konosu, S. Tetrahedron
Lett. 1985, 26, 855-856. (d) Ryu, G.; Matsunaga, S.; Fusetani, N.
Tetrahedron, 1994, 50, 13409-13416. (e) Ryu, G.; Matsunaga, S.; Fusetani,
N. Tetrahedron Lett. 1994, 35, 8251-8254.
(37) Li, H.; Matsunaga, S.; Fusetani, N. J. Nat. Prod. 1996, 59, 163-166.
(38) (a) Talpir, R.; Benayahu, Y.; Kashman, Y.; Pannell, L.; Schleyer, M.
Tetrahedron Lett. 1994, 35, 4453-4456. (b) Coleman, J. E.; de Silva, E.
D.; Kong, F.; Andersen, R. J.; Allen, T. M. Tetrahedron 1995, 51, 10653-
10662. (c) Gamble, W. R.; Durso, N. A.; Fuller, R. W.; Westergaad, C.
K.; Johnson, T. R.; Sackett, D. L.; Hamel, E.; Cardellina II, J. H.; Boyd,
M. R. Bioorg. Med. Chem. Lett. 1999, 7, 1611-1615.
(31) Freskos, J. N. Syn. Commun. 1994, 24, 557-563.
(32) (a) Yajima, H.; Fujii, N.; Funakoshi, S.; Watanabe, T.; Murayama, E.; Otake,
A. Tetrahedron 1988, 44, 805-819. (b) Nicolas, E.; Vilaseca, M.; Giralt,
E. Tetrahedron 1995, 51, 5701-5710.
(39) Thomas, B. A.; Mcmahon L. P.; Klotz, A. Z. Biochemistry 1995, 34, 3758-
3770.
9
114 J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005

Polytheonamides, Highly Cytotoxic Polypeptides
A R T I C L E S
Chart 1. Structure of Polytheonamides A and B
Avance 600 or an Avance 800 spectrometer, and data processing was
performed with XWINNMR software (Bruker) running on Silicon
Graphics O2 workstations. The DQF-COSY spectra were measured
in phase-sensitive mode. The HOHAHA spectra were recorded with a
mixing time of 66 ms. The NOESY spectra, with a mixing time of
400 ms, were measured at 300, 313, 323, and 333 K. HMBC and
HMQC spectra were recorded essentially as described in the literature.
mations, γ-N-methylasparagine and γ-N-methyl-â-hydroxyas-
paragine have not been described as components of natural
nonribosomal peptides.40
Polytheonamides are by far the largest nonribosomal peptides
and their D/L alternating stereochemistry throughout the chain
is only preceded by gramicidin A, a linear 15-residue peptide
produced by Bacillus breVis.41 This peptide is known to form
monovalent-cation selective ion channels in lipid bilayers42 and
inhibit bacterial RNA polymerase and sporulation.43 The modes
of action of polytheonamides are currently under investigation.
The origin of several nonribosomal peptides in lithistid sponges
have been traced to symbiotic bacteria.44 Perhaps, the polythe-
onamides are also derived from this source, judging from their
highly unusual structural features.
Biological Materials. The sponge samples were collected several
times by hand using Scuba at depths of 10-20 m off Hachijo-jima
Island (33° 06′ N; 139° 47′ E) and identified as Theonella swinhoei by
Dr. R. W. M. van Soest of the University of Amsterdam.
Extraction and Isolation. The frozen sponge specimens (22 kg)
were homogenized and extracted with EtOH (7 × 18 L). The combined
EtOH extracts were concentrated in vacuo and partitioned between H2O
(3 L) and diethyl ether (15 L). The ether extract was evaporated to
afford a brown gum (120 g) which was partitioned between MeOH/
H2O (9:1) and n-hexane. The aq MeOH layer (19.3 g) was subjected
to ODS flash chromatography with MeOH/H2O and MeOH/CHCl3
systems. The fraction eluted with CHCl3/MeOH (1:1) was gel-filtered
on Sephadex LH-20 with CHCl3/MeOH (3:2). Fractions (204 mg) eluted
at the column void volume were collected and further purified by ODS
HPLC on Cosmosil 5C18AR with 45% n-propanol containing 0.05%TFA
Experimental Section
General Procedures. FAB and ESI mass spectra were acquired on
a JEOL JMX-SX102/SX102 tandem mass spectrometer. Amino acid
analyses were carried out with a Hitachi 835 amino acid analyzer. For
NMR spectroscopy peptides are dissolved at concentrations of 1 mM
and 4 mM in DMSO-d6. NMR spectra were recorded on a Bruker
to furnish polytheonamides A, B, and C (3.8, 5.2, and 1.0 × 10-4
%
based on wet weight, respectively).
(40) (a) Wagatsuma, M.; Seto, M.; Miyagishima, T.; Kawazu, M.; Yamaguchi,
T.; Ohshima, S. J. Antibiot. 1983, 36, 147-154. (b) Olsufyeva, E. N.;
Berdnikova, T. F.; Miroshnikova, O. V.; Reznikova, M. I.; Preobrazhen-
skaya, M. N. J. Antibiot. 1999, 52, 319-324. (c) Faulstich, H.; Brodner,
O.; Walch, S.; Wieland, T. Justus Liebigs Ann. Chem. 1975, 2324-2330.
(d) Zhang, Y. Z.; Sun, X. C.; Zeckner, D. J.; Sachs, R. K.; Current, W. L.;
Chen, S. H. Bioorg. Med. Chem. Lett. 2001, 11, 123-126.
(41) Sarges, R.; Witkop, B. J. Am. Chem. Soc. 1965, 87, 2011-2020.
(42) Andersen, O. S.; Koeppe, R. E. Physiol. ReV. 1992, 72, S89-S158.
(43) Paulus, H.; Sarkar, N.; Mukherjee, P. K.; Langley, D.; Ivanov, V. T.; Shepel,
E. N.; Veatch, W. Biochemistry 1979, 18, 4532-4536.
(44) (a) Matsunaga, S; Fusetani, N. Curr. Org. Chem. 2003, 7, 945-966, (b)
Fusetani, N.; Matsunaga, S. Chem. ReV. 1993, 93, 1793-1806, (c) Bewley,
C. A.; Faulkner, D. J. Angew. Chem. Int. Ed. 1998, 37, 2162-2178.
Polytheonamide A: colorless amorphous solid; C219H376N60O72S;
1
FABMS m/z 5033 (M+H)+, 5055 (M+Na)+; H NMR (DMSO-d6),
see Table 3.
Polytheonamide B: colorless amorphous solid; C219H376N60O72S;
1
FABMS m/z 5033 (M+H)+, 5055 (M+Na)+; H NMR (DMSO-d6),
see Table 1. Elemental analysis for sulfur indicated the presence of
0.59% S (calcd for 0.64%).
Amino Acid Analysis of Polytheonamides. A 100 µg portion of
the peptide was dissolved in 1.0 mL of 6N HCl in an evacuated sealed
9
J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005 115

A R T I C L E S
Hamada et al.
Table 2. Amino Acid Sequence of Peptide Fragments
L-t-Leu (10.6), L-Val (11.4), L-Thr (12.3), L-Ile (14.0), D-aThr (14.9),
D-Asp (20.4), L-Asp (20.8), D-OHVal (23.7), L-OHVal (24.6), and L-Glu
(25.9).
fraction
no.
FAB−MS
(pos., m/z)
sequence
Marfey Analysis. To 2.5 µmol of the hydrolysate of peptide was
added 100 µL of 1 M NaHCO3 followed by 2.8 µmol of FDAA in 50
µL of acetone. The mixture was heated at 80 °C for 3 min. After cooling
to room temperature and the addition of 50 µL of 2N HCl, the reaction
mixture was analyzed by HPLC on Cosmosil 5C18 MS (0.5 × 25 cm):
a linear gradient from 0.05% TFA to MeCN/H2O/TFA (50:50:0.05) in
70 min at a flow rate of 1.0 mL/min: detection was with UV absorption
at 340 nm. Each peak was identified by comparing the retention time
with those of standard derivatives. The amino acid standards showed
the following retention times (min): D-OHAsp (12.2), D-Ser (16.5),
L-Thr (17.2), L-Asp (18.1), D-Asp, D-aThr (19.1), L-Glu (20.6), L-Ala
(22.4), D-Ala (26.2), L-Val (30.2), L-t-Leu (34.9), L-Ile (35.3), L-âMeIle
(39.5), and D-t-Leu (40.7).
10
19
23
361
233
332
561
702
274
602
730
659
430
602
690
589
731
589
518
717
774
602
674
661
717
830
645
774
958
401
344
1028
429
585
656
671
457
542
698
726
726
953
1024
967
811
967
910
882
754
1052
1052
995
938
995
995
1066
Val36 - OHAsm37-COOEt
Ser41 - Val42-COOEt
Ala10 - t-Leu13-COOH
Ile28 - Gly32-COOH
Asm33 - Ala38-COOH
Ala12 - Ala14-COOH
Ile34 - Ala38-COOEt
Gly19 - Gly25-COOEt
Asm33 - OHAsm37-COOEt
Ala12 - Asm15-COOEt
Ala12 - Gly17-COOEt
Asm27 - Gly32-COOH
Ile28 - Gly32-COOEt
Asm33 - Ala38-COOEt
Ile28 - Gly32-COOEt
Asm27 - t-Leu30-COOH
Asm27 - Gly32-COOEt
Gly26 - Gly32-COOEt
Ala12 - Gly17-COOEt
Ala12 - Ala18-COOEt
Asm27 - OHVal31-COOEt
Asm27 - Gly32-COOEt
Asm33 - Asm39-COOH
Ala18 - OHVal23-COOH
Ala18 - Gly25-COOH
Asm33 - Val40-COOEt
t-Leu8 - Gly11-COOEt
t-Leu8 - Ala10-COOEt
Ala7 - Gly17-COOEt
t-Leu4 - Ala7-COOH
t-Leu6 - Gly11-COOEt
t-Leu8 - Ala14-COOEt
Gly1 - Ala7-COOH
25
28
29
30
31
32
33
Partial Acid Hydrolysis. A solution of 10 mg of polytheonamide
B in 2 mL each of EtOH and 4N HCl was heated at 70 °C for 2 h in
a sealed tube. After cooling to room temperature, the solution was
evaporated to dryness under reduced pressure. The product was
dissolved in 0.5 mL of DMSO and subjected to ODS HPLC (column
size, 0.5 × 25 cm; flow rate 1.5 mL/min; first eluted with 0.05% TFA
for 20 min, and using a linear gradient from 0% to 40% MeCN in
0.05% TFA in 30 min followed by a linear gradient from 40% to 80%
MeCN in 0.05% TFA in 20 min). One-hundred twenty fractions were
obtained and analyzed by FABMS (pos.) and FABMS/MS.
36
38
39
40
42
45
46
47
52
59
60
61
65
69
72
74
80
89
90
92
93
94
95
96
97
98
100
Determination of the Chirality of the N-Terminal Residue. To a
fragment peptide (ca. 500 nM) were added 0.3 mL of 0.1M NaHCO3-
Na2CO3 buffer (pH 8.9) and 10 µM dansyl chloride-acetone solution
(0.3 mL). After stirring for 6 min at 70 °C, the solvent was removed
under reduced pressure. The product was dissolved in a small amount
of DMSO and separated by ODS HPLC (Cosmosil 5C18 MS; 0.5 × 25
cm column; linear gradient from MeCN/H2O/TFA (20:80:0.1) to
MeCN/H2O/TFA (80:20:0.1) in 50 min; 1.5 mL/min; UV detection at
340 nm). The dansylated peptide was analyzed by FAB MS and
subjected to total acid hydrolysis (6N HCl, 110 °C, 24 h) to liberate a
dansylated amino acid, which was analyzed by chiral HPLC [Sumichiral
OA-3200, 0.5 × 25 cm; elution with a solution of 0.01% AcONH4 in
MeOH (1 mL/min); detection was with UV absorption at 254 nm].
Each peak was identified by comparing the retention time with authentic
samples.
t-Leu4 - Ala7-COOEt
t-Leu4 - t-Leu9-COOH
Gly1 - Ala7-OEt
t-Leu4 - Ala10-COOH
t-Leu4 - Ala10-COOH
t-Leu4 - Ala12-COOH
Gly1 - Gly11-COOH
âMeIle2 - Gly11-COOH
t-Leu4 - Gly11-COOEt
Gly1 - Ala10-COOH
âMeIle2 - Ala10-COOH
t-Leu4 - Ala12-COOEt
t-Leu4 - Ala10-COOEt
Gly1 - Gly11-COOEt
Gly1 - Gly11-COOEt
Gly1 - Ala10-COOEt
âMeIle2 - Ala10-COOEt
Gly1 - Ala10-COOEt
Gly1 - Ala10-COOEt
âMeIle2 - Ala12-COOEt
Edman Degradation. A peptide (ca. 100 µg) was dissolved in 10
µL of EtOH/triethylamine/H2O/phenyl isothiocyanate (7:1:1:1), and
allowed to stand for 30 min at 50 °C under a nitrogen atmosphere.
The reaction mixture was centrifuged after addition of water (2.5 µL);
the suspension was washed three times with n-heptane/EtOAc (15:1)
and 4 times with n-heptane/EtOAc (1:1). The organic phases, containing
excess reagent, were discarded and the aqueous phase was evaporated
to dryness. The phenylthiocarbamylpeptide thus obtained was treated
with neat TFA (10 µL) under a nitrogen atmosphere at 50 °C for 7
min. The TFA was removed in vacuo, and the residue was kept at 50
°C for 2 min in MeOH (100 µL). After concentration, the residue was
mixed with 4 µL of H2O and 40 µL of benzene/MeCN (2:1) and
centrifuged. The lower layer containing the truncated peptide was
evaporated to dryness and subjected to dansylation, as described above.
101
102
103
104
105
tube and heated at 110 °C for 20 h. After evaporation, the residue was
dissolved in 0.4 mL of 0.01N HCl and subjected to amino acid analysis.
Retention times in the amino acid analysis of the hydrolysate of
polytheonamide B (min): OHAsp (7.4), Asp (19.4), Thr (26.8), Ser
(28.7), Glu (32.2), Gly (57.1), Ala (62.9), Val (81.0), t-Leu (88.9), Ile
(96.6), âMeIle (100.5), NH3 (142.8), MeNH2 (153.1).
Reduction of Polytheonamide B with NaBH4. To a 1.5 mL solution
of polytheonamide B (20 mg) in EtOH was added NaBH4 (5 mg), and
the solution was stirred at room temperature for 1 h. After dilution
with 5% AcOH in H2O, the solution was subjected to reversed-phase
HPLC with n-PrOH/H2O/TFA (40:60:0.05) to afford dihydropolythe-
onamide B1 (4.6 mg) and dihydropolytheonamide B2 (2.5 mg). For
the NMR spectra of these peptides, see Supporting Information.
Chiral GCMS Analysis. Chiral GC-MS was carried out using a
Chirasil L-Val capillary column (0.25 mm × 25 m) with a JEOL DX303
mass spectrometer operating in the positive EI mode (scan range
between m/z 50 and 600 with repetition times of 2 s). The acid
hydrolysate of a peptide (500 µg) was treated with 2N HCl in i-PrOH
at 90 °C for 1h in a sealed tube. After drying in vacuo, to the residue
were added CH2Cl2 and trifluoroacetic anhydride (0.3 mL, each), and
the mixture heated at 100 °C for 1 h. The reaction mixture was dried
in vacuo, and the residue was dissolved in CH2Cl2 and analyzed by
GCMS. Retention times (min): D-Ala (8.0), L-Ala (9.3), D-t-Leu (10.0),
des-Thr48-dihydropolytheonamide B2. During purification of
dihydropolytheonamide B2 by HPLC mentioned above, ca. 30% of
the peptide had been converted to des-Thr48-dihydropolytheonamide
9
116 J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005

Polytheonamides, Highly Cytotoxic Polypeptides
A R T I C L E S
Scheme 1. Reactions for stereochemical assignment of Me2Met(O)
Table 3. 1H NMR Chemical Shift of Polytheonamide A in
The retention times of all amino acid derivatives were identical with
those mentioned above. GCMS of D-Asp: m/z 255 [M+ - 59 (OC3H7)],
227 [M+ - 87 (CO2C3H7)], 213 [M+ - 101 (CO2C3H7 + CH3 -1)],
185 [M+ - 129 (CO2C3H7 + C3H7 -1)], 140 [M+ - 174 (2 ×
CO2C3H7)], 69 [(CF3) +]. GCMS of L-Asp: m/z 228 [M+ - 87
(CO2C3H7)], 214 [M+ - 101 (CO2C3H7 + CH3 -1)], 186 [M+ - 129
(CO2C3H7 + C3H7 -1)], 141 [M+ - 174 (2 × CO2C3H7)], 69 [(CF3)+].
DMSO-d6 at 313Ka
residue
NH
CRH
CâH
others
Gly1
âMeIle2
8.54 3.80
7.81 4.28
CγH30.85; CγH30.85;
CγH21.23; CδH30.82
Gly3
8.29 3.60, 3.77
7.53 4.57
8.05 4.16
7.32 4.22
7.95 4.44
7.64 4.42
7.83 4.16
8.12 4.28
8.09 3.63, 3.76
7.90 4.43
7.73 4.24
8.20 4.24
8.14 4.63
7.70 4.18
8.08 3.72
8.00 4.21
8.23 3.61, 3.88
7.65 4.10
8.32 4.59
7.72 4.28
t-Leu4
t-Leu5
t-Leu6
Ala7
t-Leu8
t-Leu9
Ala10
Gly11
Ala12
t-Leu13
Ala14
Asm15
OHVal16
Gly17
Ala18
Gly19
t-Leu20
Asm21
âMeGln22
CγH30.85
CγH30.89
CγH30.82
Reduction of Polytheonamides A and B with NH4I/Me2S. To a
solution of polytheonamide B in TFA was added NH4I (0.8 mg) and
Me2S (2 µL) and the mixture kept at 0 °C for 1h and 4 °C overnight.
After addition of 10 mg of NH4OAc, the mixture was diluted with
H2O and subjected to ODS column chromatography. Inorganic salts
were eluted with n-PrOH/H2O (2:8) and the peptides were eluted with
n-PrOH/H2O (3:1). The latter fraction was further purified by ODS
HPLC with n-PrOH/H2O/AcOH (4:6:0.2) to afford the sulfide [FABMS
(positive, nitrobenzyl alcohol with CsI), m/z 5151 (M + Cs)+, 5282
(M + 2Cs-H)+]. Polytheonamide A (2 mg) was similarly processed
to afford a peak indistinguishable from the one prepared from
polytheonamide B.
1.17
CγH30.84
CγH30.87
1.18
1.18
CγH30.84
1.14
2.36, 2.50
1.04, 1.08
1.18
BrCN treatment of the sulfide. To a solution of the above-
mentioned sulfide in HCO2H/H2O (7:3) was added BrCN (7.8 mg),
and the mixture kept at room temperature overnight in the dark. The
reaction mixture was evaporated and subjected to ODS HPLC with
n-PrOH/H2O/AcOH (45:55:2) to afford a single peak [FABMS (posi-
tive, nitrobenzyl alcohol with CsI), m/z 4657 (M+Cs)+].
CγH30.83
2.35, 2.50
2.29
CγH30.77; CγH21.79, 2.09;
NH2 6.71, 7.22
OHVal23
Ala24
Gly25
Gly26
Asm27
Ile28
8.02 4.24
7.87 4.31
8.21 3.71
8.04 3.72
8.03 4.65
7.67 4.26
CγH31.05; CγH31.12
1.18
Oxidation of Polytheonamides. To a solution of polytheonamide
B (2 mg) in n-PrOH/H2O (3:1, 0.5 mL) was added Oxone (5 mg in
100 µL of H2O) and the mixrue left at room temperature for 2 h. The
solution was diluted with H2O and subjected to ODS HPLC with
n-PrOH/H2O/AcOH (45:55:2) to afford a single peak [FABMS (nega-
tive, nitrobenzyl alcohol with CsI), m/z 5047 (M - H)-, 5308 (M +
CsI-H)-] which eluted between polytheonamides A and B. Polythe-
onamide A was processed in the same way to afford a product
indistinguishable from the one prepared from polytheonamide B in
2.36, 2.45
1.65
CγH30.96; CγH21.29;
CδH30.69
OHAsm29
t-Leu30
OHVal31
Gly32
Asm33
Ile34
7.96 4.63
7.24 4.44
8.02 4.27
8.10 3.63, 3.81
8.06 4.62
7.61 4.10
4.35
CγH30.83
CγH31.12; CγH31.12
2.37, 2.45
1.65
CγH30.98; CγH21.32;
CδH30.70
1
HPLC, MS, and H NMR data (Figure S53).
Asm35
Val36
OHAsm37
Ala38
Asm39
Val40
Ser41
8.17 4.65
7.64 4.06
7.98 4.62
7.68 4.21
8.01 4.62
7.62 4.21
8.05 4.40
7.80 4.21
8.24 4.63
2.35, 2.50
1.87
4.39
1.18
2.38, 2.51
1.97
CγH30.72; CγH30.72
Acknowledgment. This work was partly supported by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science, and Technology. We thank
Professors T. Kitahara and H. Watanabe of University of Tokyo
for helpful discussions in amino acid synthesis. Thanks are also
due to Dr. K. Drauz and Dr. A. Bommarius of Degussa and
Dr. N. Imamura of University of Ritsumeikan for gifts of amino
acids. JSPS Research Fellowships to T.H. are greatly acknowl-
edged. Finally, we are indebted to an anonymous referee for
improving our English very much.
CγH30.76; CγH30.76
CγH30.78; CγH30.78
3.51, 3.56
1.93
Val42
Asn43
2.41, 2.52 NH2 6.85, 7.25
CγH31.02; CγH31.08;
CγH22.75, 2.85; CꢀH32.60
2.42, 2.54 NH2 6.85, 7.31
Me2Met(O)44 7.91 4.24
Asn45
Gln46
8.35 4.54
7.86 4.29
2.06
CγH21.74, 1.88;
NH2 6.65, 7.11
CγH31.03
aThr 47
Thr48
7.99 4.31
7.68 4.15
3.86
4.08
CγH31.02
Supporting Information Available: (1) spectral data and 13
C
a N-methyl amides in the side chain: NH/CH3, 7.62/2.55, 7.63/2.55, 7.65/
2.54, 7.66/2.52, 7.71/2.54, 7.72/2.54, 7.75/2.56, 7.79/2.55.
NMR chemical shift Table of polytheonamides B, (2) spectral
data of polytheonamides A, (3) amino acid analysis, elemental
1
B2 [FABMS (negative mode, triethanolamine) m/z 4935 (M-H)-]
analysis, spectral data and H NMR chemical shift Table of
acid hydrolysate of polytheonamide B, (4) spectral data of
dihydropolytheonamide B1, (5) chiral GCMS data of trifluo-
roacetyl isopropyl ester derivatives and HPLC charts of FDAA
derivatives of polytheonamide B, (6) FABMS/MS spectra of
which eluted faster than dihydropolytheonamide B2.
Hydrolysis with DCl/D2O. A 200 µg portion of the peptide was
hydrolyzed with 6N DCl at 110 °C for 10 h. The acid hydrolysate was
processed in the same way as mentioned for the chiral GCMS analysis.
9
J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005 117

A R T I C L E S
Hamada et al.
fragment peptides of polytheonamide B by the partial acid
hydrolysis [4N HCl/EtOH (1:1), 70 °C, 30 min.], (7) N-terminal
analysis, Marfey analysis and chiral GCMS data for determining
chiralities of amino acid residues in polytheonamide B, (8)
Experimental Section describing synthesis of âMeGlu, (9)
spectral data of des-Thr48-dihydropolytheonamide B2, (10)
GCMS data of D- and L-Asp derivatives after DCl hydrolysis,
(11) spectral data for determining the stereochemistry of
Me2Met(O)44 in polytheonamide B, (12) spectral data of the
oxidized products of polytheonamides A and B by Oxone (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA045749E
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118 J. AM. CHEM. SOC. VOL. 127, NO. 1, 2005