Tamandarins A and B: New cytotoxic depsipeptides from a Brazilian ascidian of the family didemnidae

Article abstract of DOI:10.1021/jo991425a

The structures of two new, naturally occurring cytotoxic depsipeptides, tamandarins A and B (1 and 2) are presented. The tamandarins were isolated from an unidentified Brazilian marine ascidian of the family Didemnidae. The structures of the new cytotoxins were assigned by interpretation of FABMS data and by extensive 2D NMR analyses. The absolute configurations of the tamandarins were assigned by acid and alkaline hydrolysis to yield their corresponding amino acids, which were then analyzed as their Marfey derivatives. The cytotoxicity of tamandarin A (1) was evaluated against various human cancer cell lines and shown to be slightly more potent than didemnin B. A qualitative discussion of the conformation of tamandarin A (1) in solution, obtained from NMR J-value data, variable temperature experiments, and NOESY/ROESY data, is included.

Full text of DOI:10.1021/jo991425a

782
J . Org. Chem. 2000, 65, 782-792
Ta m a n d a r in s A a n d B: New Cytotoxic Dep sip ep tid es fr om a
Br a zilia n Ascid ia n of th e F a m ily Did em n id a e
He´le`ne Vervoort and William Fenical*
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of
CaliforniasSan Diego, La J olla, California 92093-0204
Rosaˆngela de A. Epifanio
Departamento de Quimica Orgaˆnica, Instituto de Qu´ımica, Universidade Federal Fluminense,
24020-150 Nitero´i, Rio de J aneiro, Brazil
Received September 9, 1999
The structures of two new, naturally occurring cytotoxic depsipeptides, tamandarins A and B (1
and 2), are presented. The tamandarins were isolated from an unidentified Brazilian marine ascidian
of the family Didemnidae. The structures of the new cytotoxins were assigned by interpretation of
FABMS data and by extensive 2D NMR analyses. The absolute configurations of the tamandarins
were assigned by acid and alkaline hydrolysis to yield their corresponding amino acids, which were
then analyzed as their Marfey derivatives. The cytotoxicity of tamandarin A (1) was evaluated
against various human cancer cell lines and shown to be slightly more potent than didemnin B. A
qualitative discussion of the conformation of tamandarin A (1) in solution, obtained from NMR
J -value data, variable temperature experiments, and NOESY/ROESY data, is included.
Marine invertebrates of the class Ascidiacea (phylum
fied didemnid8 ascidian on a shallow-water reef near the
Brazilian village of Tamandare´. The crude extract of this
animal showed potent growth inhibitory activity in our
primary cytotoxicity assay employing the human colon
tumor cell line HCT 116. Subsequent bioassay-guided
fractionation using LH-20 column chromatography and
RP (C18) HPLC yielded two cytotoxic depsipeptides, the
major metabolite tamandarin A (1) and a minor metabo-
lite, tamandarin B (2, Chart 1).
Analysis of spectral data revealed that tamandarins
A (1) and B (2) are closely related to didemnin B and
nordidemnin B, respectively. The molecules were found
to differ only by the presence of hydroxyisovaleric acid
(Hiv2), instead of the hydroxyisovalerylpropionic acid
(Hip2) unit which is present in all other naturally
occurring didemnin congeners. Tamandarin B (2) was
found to contain a norstatine (Nst1) residue instead of
the isostatine (Ist1) residue of tamandarin A (1). In this
paper, we report the isolation and structure elucidation
of tamandarins A (1) and B (2) and provide preliminary
pharmacological information on tamandarin A (1). A
Chordata, subphylum Urochordata) have emerged as a
rich source of nitrogen-containing metabolites that often
exhibit potent anticancer activities. Studies of the Carib-
bean mangrove ascidian Ecteinascidia turbinata, whose
potent anticancer activities were first discovered by Sigel
et al.,1 led to the isolation of ecteinascidin 743, a drug
candidate currently in clinical trials. Ecteinascidin 743
showed significant activities against murine (L1210)
leukemia in vitro (IC50) 0.5 ng/mL) and in vivo activities
against P-388 lymphocytic leukemia and human mam-
mary tumors. Chemical studies of another Caribbean
ascidian, Trididemnum solidum, led to the discovery of
the didemnin depsipeptides, of which didemnin B is the
most well-known member.3,4 Although didemnin B showed
significant in vivo activity in mice,3,5 human clinical
testing showed didemnin B to be toxic at doses near those
required for therapeutic applications. Ascidians of the
genus Lissoclinum have also been prolific sources of
cytotoxic secondary metabolites such as patellamide A
and ulithiacyclamide.6,7
As part of our program to explore marine ascidians for
their applications in cancer, we encountered an unidenti-
(4) (a) Guyot, M.; Davoust, D.; Morel, E. C. R. Acad. Sci. Paris Ser.
II 1987, 305, 681-686. (b) Gloer, J . B. Ph.D. Dissertation, University
of Illinois, Urbana, IL, 1983; Chem. Abstr. 1983, 103, 122692b; Diss.
Abstr. Int. B 1983, 45, 188-189. (c) Gutowsky, R. E. M.S. Thesis,
University of Illinois, Urbana, IL, 1984. (d) Sakai, R.; Stroh, J . G.;
Sullins, D. W.; Rinehart, K. L., J r. J . Am. Chem. Soc. 1995, 117, 3734-
3748. (e) Boulanger, A.; Abou-Mansour, E.; Badre, A.; Banaigs, B.;
Combaut, G.; Francisco, C. Tetrahedron Lett. 1994, 35, 4345-4348.
(f) Rinehart, K. L. J r. British Patent Appl. 8922026.3, Sept 29, 1989.
(g) Abou-Mansour, E.; Boulanger, A.; Badre, A.; Bonnard, I.; Banaigs,
B.; Combaut, G.; Francisco, C. Tetrahedron 1995, 51, 12591-12600.
(5) Fimiani, V. Oncology 1987, 44, 42-46.
(1) (a) Sigel, M. M.; Wellham, L. L.; Lichter, W.; Dudeck, L. E.;
Gargus, J . L.; Lucas, A. H. In Food-Drugs from the Sea Proceedings
1969; Younghen, H. W., J r., Ed.; Marine Technol. Soc.: Washington,
DC, 1970; pp 281-294. (b) Lichter, W.; Ghaffar, A.; Wellham, L. L.;
Sigel, M. M. In Drugs & Food from the Sea; Kaul, P. N., Sindermann,
C. J ., Eds.; University of Oklahoma: Norman, OK, 1978; pp 137-144.
(c) Sigel, M. M.; Lichter, W.; Wellham, L. L.; McCumber, L. J . 4th
International Symposium on Marine Natural Products, Tenerife,
Spain, J uly 26-30, 1982; Abstract C-12.
(2) (a) Holt, T. G. Ph.D. Dissertation, University of Illinois, Urbana,
1986; Chem. Abstr. 1987, 106, 193149u; Diss. Abstr. Int. B 1987, 47,
3771-3772. (b) Sakai, R.; Rinehart, K. L.; Guan, Y.; Wang, A. H.-J .
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11456-11460.
(3) (a) Rinehart, K. L., J r.; Gloer, J . B.; Hughes, R. G.; Renis, H. E.;
McGovren, J . P.; Swynenberg, E. B.; Stringfellow, D. A.; Kuentzel, S.
L.; Li, L. H. Science 1981, 212, 933-935. (b) Rinehart, K. L., J r.; Gloer,
J . B.; Cook, J . C. J r.; Mizsak, S. A.; Scahill, T. A. J . Am. Chem. Soc.
1981, 103, 1857-1859. (c) Rinehart, K. L., J r. U.S. Patent 4,493,796,
J an 15, 1985; Chem. Abstr. 1985, 103, 76241v. (d) Rinehart, K. L., J r.
U.S. Patent 4,548,814, Oct 22, 1985.
(6) (a) Ireland, C. M.; Scheuer, P. J . J . Am. Chem. Soc. 1980, 102,
5688-5691. (b) Ireland, C. M.; Durso, A. R., J r.; Newman, R. A.;
Hacker, M. P. J . Org. Chem. 1982, 47, 1807-1811. (c) Biskupiak, J .
E.; Ireland, C. M. J . Org. Chem. 1983, 48, 2302-2304.
(7) (a) Schmitz, F. J .; Ksebati, M. B.; Chang, J . S.; Wang, J . L.;
Hossain, M. B.; van der Helm, D.; Engel, M. H.; Serban, A.; Silfer, J .
A. J . Org. Chem. 1989, 54, 3463-3472. (b) McDonald, L. A.; Ireland,
C. M. J . Nat. Prod. 1992, 55, 376-379.
(8) Identification as family Didemnidae was carried out by Dr.
Franc¸oise Monniot, Laboratoire de Biologie des Inverte´bre´s Marins et
Malacology, Muse´um Nationale d’Histoire Naturelle, Paris, France.
10.1021/jo991425a CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/29/1999

Tamandarins A and B: New Cytotoxic Depsipeptides
J . Org. Chem., Vol. 65, No. 3, 2000 783
Ch a r t 1
F igu r e 1. Structure of tamandarin A (1) showing fragment
ions obtained by MS/MS (m/z 1057 [M+ + H]).
carbon resonances were all in the aliphatic region. DEPT
and HMQC experiments established the multiplicities of
each carbon and all one-bond 1H-13C correlations, in
agreement with the molecular formula. The spin systems
of Lac9, Pro4, and Pro8 units, a leucine (Leu3) and MeLeu7
unit, a threonine (Thr6) unit, and parts of the Ist1 and
N,O-dimethyltyrosine (Me2Tyr5) were identified by cor-
relations observed in a double-quantum filtered COSY
experiment (DQF-COSY). In addition, a Hiv2 unit was
identified, but the propionic acid of Hip2 of didemnin B
was clearly absent in tamandarin A.
As was pointed out by Kessler et al. for didemnin B,
several correlations within Ist1 (C5H to C6H) and Leu3
and MeLeu7 (CâH to CγH) do not appear in the COSY
spectrum.9 These correlations were not observed for
tamandarin A (1), but they were obtained from a TOCSY
experiment which also confirmed the proposed units.
A ROESY experiment established the sequence of the
amino acids and confirmed the absence of the propionic
acid in the Hip2 unit. An HMBC experiment provided
sequence data and allowed all carbonyl functionalities,
except for the Leu3 CO carbon, to be assigned. This
experiment also confirmed the ester bonds between the
Ist1 and Hiv2 units, and between the Thr6 and Me2Tyr5
units. On the basis of these data, tamandarin A (1) was
assigned with confidence as possessing a hydroxyisova-
leric acid (Hiv2) unit instead of the hydroxyisovaleryl-
propionic acid (Hip2) unit found in didemnin B.
Hydrolysis of tamandarin A (1) under mild alkaline
conditions (1 N NaOH in MeOH, 1 h, rt, Chart 2) yielded
the northern and southern peptide fragments 3 and 4,
which were converted to their methyl esters by reaction
with diazomethane. In a similar reaction, didemnin B
was hydrolyzed to yield the analogous peptide fragments.
Comparison of the 1H NMR spectra, optical rotations, and
tandem mass spectral results of the tamandarin A
peptides with those obtained from didemnin B10,11 re-
vealed that the northern fragments, 3, from both peptides
are identical ([R]D ) +41°, c ) 0.07). The chiralities of
the amino acid residues in the northern fragment (3)
were thus established as (S)-Lac9, L-Pro8, D-MeLeu7,
L-Thr6, and (3S,4R,5S)-Ist1. The chiralities of the amino
acids constituting the southern peptide fragment (4) of
tamandarin A (1) were obtained by acid-catalyzed hy-
drolysis, preparation of the corresponding Marfey’s de-
comparison of the conformation of this metabolite in
solution versus that of didemnin B is also presented.
Tamandarin A (1) was obtained as a greenish white
solid. Mass spectral analysis (FABMS) of this metabolite
showed a pseudomolecular ion consistent with the mo-
1
lecular formula C54H87N7O14. The H NMR spectrum of
tamandarin A (1, Table 1) showed several characteristic
features that strongly resembled those of didemnin B.
Three NH protons were visible in the amide hydrogen
region (δ 7.3-7.8 ppm), and a para-substituted phenyl
ring was apparent (δ 7.07, d, 2H, J ) 8.4 Hz; δ 6.84, d,
2H, J ) 8.7 Hz). However, only 10 well-resolved apparent
R-hydrogens were visible (δ 3.8-5.5 ppm), whereas
didemnin B possesses 11. Three methyl groups on het-
eroatoms (δ 3.79, s, 3H, OCH3; δ 3.11, s, 3H, NCH3; δ
2.58, s, 3H, NCH3) were observed, but only 10 additional
methyl groups could be accounted for (δ 0.8-1.5 ppm),
while didemnin B has 11. These findings indicated that
tamandarin A (1) is a novel didemnin congener. Tandem
mass spectral analysis (MS/MS, Figure 1) was under-
taken to establish the relationship between these two
molecules. The fragmentation pattern of the parent ion
at 1056 amu (M + H+) showed the loss of a lactic acid
(Lac9) and a proline (Pro8) as one unit, 169 (m/z 887), and
loss of 127 (m/z 760) for an N-methylleucine (MeLeu7)
unit, indicating that tamandarin A (1) possesses the same
side chain as didemnin B. Hence, the noted modifications
must be within the macrocyclic ring. The 13C NMR
spectrum of tamandarin A (1) showed 54 carbon reso-
nances, including nine carbonyl carbons (δ 168-175
ppm). Unlike didemnin B, tamandarin A did not possess
a ketone, the carbonyl shift of the former being observed
at approximately δ 200 ppm. Six carbon resonances
appeared in the aromatic region, with the phenyl ring
carbons overlapping (δ 114-160 ppm). The remaining 39
(9) (a) Kessler, H.; Will, M.; Sheldrick, G. M.; Antel, J . Magn. Reson.
Chem. 1988, 26, 501-506. (b) Kessler, H. Will, M.; Antel, J .; Beck, H.;
Sheldrick, G. M. Helv. Chim. Acta 1989, 72, 530-555.
(10) Didemnin B was kindly provided by Dr. D. J . Faulkner.
(11) Banaigs, B.; J eanty, G.; Francisco, C.; J ouin, P.; Poncet, J .;
Heitz, A.; Cave, A.; Prome, J . C.; Wahl, M.; Lafargue, F. Tetrahedron
1989, 45, 181-190.

784 J . Org. Chem., Vol. 65, No. 3, 2000
Ta ble 1. NMR Assign m en ts for Ta m a n d a r in A (1)
Vervoort et al.
gHMBC
structural unit
position
13C (CDCl3) δ (mult)a
1H (CDCl3) δ (mult, J , integ)a
COSY
(3S,4R,5S)-Ist1
1
2
174.7 (C)
39.9 (CH2)
5.03, 3.25, 2.44
4.03
(a) 2.44 (dd, 17.0, 8.1 Hz, 1H)
(b) 3.25 (br d, 17.0 Hz, 1H)
3.91 (br tr, 9.6, 8.1 Hz, 1H)
n.o.b
3.25, 3.91
2.44, 3.91
2.44, 3.25
3
69.1 (CH)
2.44,
3-OH
4
5
6
55.4 (CH)
33.8 (CH)
27.6 (CH2)
4.03 (tr d, 9.6, 9.6, 3.4 Hz, 1H)
1.96 (m, 1H)
1.20 (dd, 13.8, 6.9 Hz, 1H)
1.40 (m, 1H)
0.92 (tr, 6.9, 6.9 Hz, 3H)
0.88 (d, 6.3 Hz, 3H)
7.36 (br d, 9.6 Hz, 1H)
7.36, 1.96
4.03, 0.88
1.40, 0.92
1.20, 0.92
1.20, 1.40
1.96
3.91, 3.25, 1.20
1.40, 1.20
7
8
9
12.0 (CH3)
14.3 (CH3)
1.40, 1.20
4.03, 1.40, 1.20
4.03
(S)-Hiv2
CO
R
â
â-CH3
â-CH3
CO
R
â
169.8 (C)
79.1 (CH)
30.3 (CH)
17.8 (CH3)
19.1 (CH3)
170.6 (C)*
48.5 (CH)
39.6 (CH2)
5.03
5.03 (d, 4.8 Hz, 1H)
2.26 (m, 1H)
1.04 (d, 6.6 Hz, 3H)
1.03 (d, 6.6 Hz, 3H)
2.26
1.03, 1.04
2.26
5.03
5.03, 2.26
5.03, 2.26
2.26
L-Leu3
4.88 (tr d, 9.6, 9.6, 1.6 Hz, 1H)
1.25 (m, 1H)
1.50 (m, 1H)
1.25, 1.50, 7.77
1.50, 4.88
1.25, 4.88
0.95, 0.91
1.55
0.95, 0.91
0.95, 0.91
γ
25.0 (CH)
21.1 (CH3)
24.0 (CH3)
1.55 (m, 1H)
γ-CH3
γ-CH3
NH
CO
R
0.95 (d, 6.3 Hz, 3H)
0.91 (d, 6.3 Hz, 3H)
7.77 (br d, 9.6 Hz, 1H)
1.55
4.88
L-Pro4
171.3 (C)
57.2 (CH)
28.2 (CH2)
2.10, 1.77
4.65, 3.65
4.65 (dd, 7.8, 3.6 Hz, 1H)
1.77 (m, 1H)
2.10 (m, 1H)
2.10 (m, 2H)
3.65 (m, 2H)
1.77, 2.10
2.10, 4.65
1.77, 4.65
3.65
â
γ
δ
CO
R
â
25.1 (CH2)
46.9 (CH2)
168.7 (C)
66.4 (CH)
34.1 (CH2)
4.65, 3.65, 2.10
1.77
5.42, 3.59, 3.13
3.40, 3.13
2.10
L-Me2Tyr5
3.59 (dd, 10.8, 4.1 Hz, 1H)
3.13 (dd, 14.6, 10.8 Hz, 1H)
3.40 (dd, 14.6, 4.1 Hz, 1H)
3.13, 3.40
3.40, 3.59
3.13, 3.59
7.07, 3.59
γ
130.3 (C)
3.59, 3.40, 3.13
3.40, 3.13
7.07
δ
130.5 (CH)
114.3 (CH)
158.8 (C)
7.07 (d, 8.6 Hz, 2H)
6.84 (d, 8.6 Hz, 2H)
6.84
7.07
ꢀ
ú
7.07, 3.79
γ-CH3
N-CH3
CO
R
55.5 (CH3)
38.9 (CH3)
170.3 (C)
58.1 (CH)
70.9 (CH)
16.7 (CH3)
3.78 (s, 3H)
2.58 (s, 3H)
L-Thr6
7.36, 4.03
5.42
7.47, 4.26, 1.35
5.42, 4.26
4.26 (dd, 5.4, 1.4 Hz, 1H)
5.42 (q d, 6.0, 1.4 Hz, 1H)
1.35 (d, 6.0 Hz, 3H)
7.47, 5.42
1.35, 4.26
5.42
â
â-CH3
NH
CO
R
7.46 (br d, 5.4 Hz, 1H)
4.26
D-MeLeu7
170.8 (C)
55.1 (CH)
35.9 (CH2)
7.47, 1.88
3.11, 1.66
5.30
5.30 (dd, 11.4, 3.6 Hz, 1H)
1.88 (m, 1H)
1.66 (m, 1H)
1.88, 1.66
1.66, 1.35, 5.30
1.88, 1.35, 5.30
0.84, 0.91
1.35
â
γ
25.0 (CH)
23.7 (CH3)
21.5 (CH3)
31.5 (CH3)
172.9 (C)
1.35 (m, 1H)
5.30
â-CH3
â-CH3
N-CH3
CO
0.91 (d, 6.9 Hz, 3H)
0.84 (d, 6.9 Hz, 3H)
3.11 (s, 3H)
1.35
1.88, 1.66
5.30
5.30, 3.11
3.59
L-Pro8
R
â
56.9 (CH)
28.6 (CH2)
4.72 (br t, 7.5, 7.5 Hz, 1H)
1.96 (m, 1H)
2.22 (m, 1H)
2.22, 1.96
2.22
1.96
4.72, 1.96
γ
δ
26.2 (CH2)
47.2 (CH2)
1.96 (m, 1H)
2.22 (m, 1H)
3.65 (m, 1H)
3.59 (dd, 10.8, 3.9 Hz, 1H)
2.22
1.96
3.65, 2.22, 1.96
3.59, 2.22, 1.96
3.59
2.22
(S)-Lac9
CO
R
R-CH3
R-OH
174.0 (C)
66.3 (CH)
20.5 (CH3)
4.38
1.43
4.38
4.38 (br q, 6.9, 1H)
1.43
4.38
1.43 (d, 6.9, 3H)
n.o.b
1H and 13C NMR shifts were referenced to CDCl3 (1H δ 7.27 and 13C δ 77.2 ppm). Not observed.
a
b
rivatives, and HPLC analysis using D- and L-amino acid
(Marfey’s derivatives) standards.12 All comparable amino
acids in this fragment were shown to have absolute
configurations identical with those in didemnin B: L-Me2-
Tyr5, L-Pro4, and L-Leu3. Acid hydrolysis of the southern
peptide fragment (4) did not cleave the methyl ester link
in L-Me2Tyr. Therefore, a mixture of the Marfey deriva-
tives of D- and L-Me2Tyr methyl ester (Me3Tyr) was
prepared and used in the HPLC analysis (Chart 2).
The absolute stereochemistry of the Hiv2 unit could not
be obtained from the Marfey derivative HPLC analysis.
Instead, the stereochemistry of the Hiv2 unit was deter-
(12) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.

Tamandarins A and B: New Cytotoxic Depsipeptides
J . Org. Chem., Vol. 65, No. 3, 2000 785
Ch a r t 2
ment with the molecular formula. COSY and TOCSY
experiments established the connectivities within the
Nst1 residue and confirmed the presence of the Lac9 unit
and the same remaining amino and hydroxy acids as in
tamandarin A. Determination of the amino acid sequence
and assignment of all carbonyl functionalities in taman-
darin B (2) was accomplished by ROESY and HMBC
NMR experiments. Tamandarin B (2) was assigned with
confidence as differing from tamandarin A (1) by the
presence of the amino acid Nst1 instead of the Ist1 unit
of tamandarin A (1).
Similar to tamandarin A, hydrolysis of 2 yielded the
northern and southern peptide fragments (3′ and 4),
which were also converted to their methyl esters with
diazomethane. Likewise, hydrolysis of nordidemnin B14
yielded peptide fragments which were compared to those
from tamandarin B. The northern peptide fragment (3′)
of tamandarin B (2) ([R]D ) +24°, c ) 0.05) was found to
be identical to the northern fragment of nordidemnin B,
thus indicating the absolute configurations of the amino
acids to be identical: (S)-Lac,9 L-Pro8, D-MeLeu7, L-Thr6,
and (3S,4R)-Nst1. The southern peptide fragment (4) from
1
tamandarin B (2) showed the same H NMR spectrum
and optical rotation as the southern peptide fragment (4)
from tamandarin A (1), indicating the chiralities of the
amino acids to be identical (L-Me2Tyr5, L-Pro4, L-Leu3, and
(S)-Hiv2).
1
mined by analysis of the H NMR spectra of the diaster-
eomeric (R)- and (S)-Mosher ester derivatives of the
southern peptide fragment (4).13 In the (S)-MTPA ester,
all protons in the Hiv2 unit were more highly shielded
(appeared further upfield), whereas the R-hydrogen and
amide hydrogens in the neighboring leucine unit were
less highly shielded (and appeared farther downfield).
The reverse was true for the (R)-MTPA ester of the
southern peptide fragment (4), permitting the identifica-
tion of the absolute stereochemistry of the hydroxyisova-
leric acid as (S)-Hiv2.
Tamandarin B (2), obtained as an amorphous white
solid, showed a FABMS pseudomolecular ion consistent
with the molecular formula C53H82N7O14. The lower mass
of this metabolite, shifted by 14 units as compared with
tamandarin A (1), suggested that the difference was the
Given the comparison of tamandarin A (1) with di-
demnin B, studies were undertaken to document the
tertiary structure of this molecule. Several independent
studies of the conformation of didemnin B showed that
it exhibits conformational constancy from crystal to
solution structure and in a range of polar and apolar
solvents.4g,9b,11,15 Each study showed the molecule to exist
in a globular shape, stabilized by at least three intramo-
lecular hydrogen bonds. The strongest hydrogen bond is
a transannular interaction linking Leu3 CO and Ist1 NH.
This gives the macrocyclic ring the shape of a distorted
“figure eight” rather than the flat antiparallel â-sheet-
type arrangement more commonly observed in cyclic
depsipeptides. The linear portion of didemnin B is
involved in a â II-type turn, a structural feature often
observed in linear peptides, in which four amino acids
are stabilized in a well-defined conformation by hydrogen
bonding between amino acids i (H donor) and i + 3 (H
acceptor). The â turn in didemnin B encompasses resi-
dues Thr6, MeLeu7, Pro8, and Lac9, with hydrogen-
bonding interaction consistently found between Thr6 NH
and Lac9 CO. The third hydrogen bond in didemnin B
was identified between MeLeu7 CO in the linear portion
of the molecule and Leu3 NH in the macrocycle, orienting
the folded linear chain back over the macrocyclic ring,
resulting in its overall globular shape.
1
absence of a methylene group. The H NMR spectrum of
tamandarin B (2, Table 2) was almost identical to that
of tamandarin A (1), except for a small downfield shift
(∆δ ) 0.2 ppm) in one of the hydrogens in the amide
region (δ 7.2-7.8 ppm) and slight upfield shifts (∆δ )
0.05 ppm) of two hydrogens in the R-hydrogen region (δ
3.8-5.5 ppm). Perhaps more importantly, the latter
protons showed a change in coupling pattern. In addition,
a hydrogen at δ 1.20 ppm, showing an obvious multiplet
1
structure in the H spectrum of tamandarin A (1), was
lacking. These observations indicated that tamandarin
B (2) contains a Nst1 residue instead of the Ist1 residue
in tamandarin A (1).
With some difficulty from overlapping bands, the
replacement of the triplet methyl signal of tamandarin
A (1) at δ 0.92 ppm with a doublet methyl in tamandarin
B (1) at δ 0.97 ppm was resolved. The 13C NMR spectrum
of tamandarin B (2) was nearly identical to that of
tamandarin A (1), except for the lack of one methylene
resonance in the aliphatic region. DEPT and HMQC
experiments confirmed the multiplicity of each carbon
To assess the effects of the ring modifications in
tamandarin A, conformational studies were undertaken
1
using J values from 1D H or 2D ECOSY NMR spectra,
NH chemical shifts and their temperature dependence
(Table 4), and NOE/ROE correlations obtained from 2D
NOESY and ROESY experiments (Table 5). An effort was
and showed all one-bond H-13C correlations in agree-
1
(14) Nordidemnin B was kindly provided by Dr. N. Lindquist.
(15) (a) Hossain, M. B.; van der Helm, D.; Antel, J .; Sheldrick, G.
M.; Sanduja, S. K.; Weinheimer, A. J . Proc. Natl. Acad. Sci. U.S.A.
1988, 85, 4118-4122. (b) Searle, M. S.; Hall, J . G.; Kyratzis, I.;
Wakelin, L. P. G. Int. J . Pept. Protein Res. 1989, 34, 445-454. (c)
Kessler, H.; Mronga, S.; Will, M.; Schmidt, U. Helv. Chim. Acta 1990,
73, 25-47.
(13) Rieser, M. J .; Hui, Y.-H.; Rupprecht, J . K.; Kozlowski, J . F.;
Wood, K. V.; McLaughlin, J . L.; Hanson, P. R.; Zhuang, Z.; Hoye, T. J .
Am. Chem. Soc. 1992, 114, 10203-10213. (b) Dale, J . A.; Mosher, H.
S. J . Am. Chem. Soc. 1973, 95, 512-519.

786 J . Org. Chem., Vol. 65, No. 3, 2000
Ta ble 2. NMR Assign m en ts for Ta m a n d a r in B (2)
Vervoort et al.
structural unit
position
13C (CDCl3) δ (mult)a
1H (CDCl3) δ (mult, J , integ)a
COSY
gHMBC
(3R,4S)-Nst1
1
2
174.2 (C)
39.7 (CH2)
3.25, 2.48
(a) 2.48 (dd, 17.0, 7.5 Hz, 1H)
(b) 3.25 (dd, 17.0, 2.7 Hz, 1H)
3.96 (m, 1H)
3.96, 3.34
3.96, 3.48
3.86, 3.25, 2.48
3.96
3
69.2 (CH)
2.48
3-OH
3.34 (m, 1H)
4
5
6
58.8 (CH)
27.3 (CH)
17.3 (CH3)
3.86 (dd, 8.8, 5.0 Hz, 1H)
2.13 (m, 1H)
0.91 (d, 6.9 Hz, 3H)
7.56, 3.96, 2.13
3.86, 0.97, 0.91
2.13
0.97, 0.91
0.97, 0.91
0.97
7
8
9
20.7 (CH3)
0.97 (d, 6.9 Hz, 3H)
7.56 (br d, 8.8 Hz, 1H)
2.13
3.86
0.91
(S)-Hiv2
CO
R
â
â-CH3
â-CH3
CO
R
â
169.9 (C)
78.9 (CH)
30.4 (CH)
17.7 (CH3)
19.2 (CH3)
171.3 (C)
48.5 (CH)
39.5 (CH2)
5.04
1.04
5.04, 1.05, 1.04
1.04
1.05
5.04 (d, 4.5 Hz, 1H)
2.25 (m, 1H)
1.05 (d, 6.9 Hz, 3H)
1.04 (d, 6.9 Hz, 3H)
2.25
1.05, 1.04
2.25
2.25
L-Leu3
4.89 (tr d, 9.8, 9.8, 1.6 Hz, 1H)
1.33 (m, 1H)
1.51 (m, 1H)
7.78
1.51
1.33
0.96, 0.92
γ
25.1 (CH)
21.7 (CH3)
24.0 (CH3)
1.59 (m, 1H)
0.96, 0.92
1.59
1.59
0.96, 0.92
0.92
0.96
γ-CH3
γ-CH3
NH
CO
R
0.96 (d, 6.9 Hz, 3H)
0.92 (d, 6.9 Hz, 3H)
7.78 (br d, 9.9 Hz, 1H)
4.89
L-Pro4
170.9 (C)
57.2 (CH)
28.2 (CH2)
3.60, 2.59, 1.84
4.65 (dd, 7.5, 4.1 Hz, 1H)
1.84 (m, 1H)
2.05 (m, 1H)
2.05 (m, 2H)
3.67 (m, 2H)
2.05, 1.84
2.05
1.84
3.67
2.05
â
4.65
4.65
γ
δ
CO
R
â
25.1 (CH2)
46.9 (CH2)
168.8 (C)
66.5 (CH)
34.2 (CH2)
L-Me2Tyr5
5.42, 3.60
3.41, 3.16, 2.59
3.60
3.60 (dd, 10.8, 4.0 Hz, 1H)
3.16 (dd, 14.1, 10.5 Hz, 1H)
3.40 (dd, 14.1, 4.0 Hz, 1H)
3.40, 3.16
3.40, 3.60
3.60, 3.16
γ
130.0 (C)
6.85, 3.60, 3.41
3.16
δ
130.6 (CH)
114.3 (CH)
158.8 (C)
7.08 (d, 8.3 Hz, 2H)
6.85 (d, 8.3 Hz, 2H)
6.85
7.08
6.85, 3.41, 3.16
6.85
7.08, 6.85, 3.80
ꢀ
ú
γ-CH3
N-CH3
CO
R
55.5 (CH3)
38.9 (CH3)
170.8 (C)
58.0 (CH)
71.0 (CH)
16.8 (CH3)
3.80 (s, 3H)
2.59 (s, 3H)
3.60
7.56
1.38
7.49, 4.31, 1.38
L-Thr6
4.31 (dd, 5.6, 1.4 Hz, 1H)
5.42 (qd, 6.5, 1.4 Hz, 1H)
1.38 (d, 6.5 Hz, 3H)
7.49, 5.42
4.32, 1.38
5.42
â
â-CH3
NH
CO
R
7.49 (br d, 5.6 Hz, 1H)
4.31
D-MeLeu7
170.7 (C)
55.1 (CH)
36.8 (CH2)
7.49
3.11
0.92, 0.85
5.31 (dd, 11.4, 3.6 Hz, 1H)
1.88 (m, 1H)
1.67 (m, 1H)
1.88, 1.67
1.67
1.88
â
γ
25.1 (CH)
1.38 (m, 1H)
1.88, 1.67
0.92, 0.85, 0.92
0.85
â-CH3
â-CH3
N-CH3
CO
23.7 (CH3)
21.6 (CH3)
31.4 (CH3)
172.9 (C)
0.92 (d, 6.9 Hz, 3H)
0.85 (d, 6.9 Hz, 3H)
3.11 (s, 3H)
1.38
1.38
0.85
1.88, 1.67, 0.92
5.31
L-Pro8
5.31, 3.11
R
â
57.0 (CH)
28.6 (CH2)
4.73 (br t, 7.8, 7.8 Hz, 1H)
1.97 (m, 1H)
2.13 (m, 1H)
2.13, 1.97
2.13
1.97
γ
δ
26.2 (CH2)
47.3 (CH2)
1.97 (m, 1H)
2.13 (m, 1H)
3.67 (m, 1H)
3.60 (dd, 10.8, 4.1 Hz, 1H)
2.13
1.97
1.97
2.13
4.73
(S)-Lac9
CO
R
R-CH3
R-OH
174.1 (C)
66.3 (CH)
20.5 (CH3)
1.44
1.44
4.39 (br q, 6.9 Hz, 1H)
1.44 (d, 6.9 Hz, 3H)
3.44 (m, 1H)
3.44, 1.44
4.39
4.39
made to identify the major differences in shape between
tamandarin A and didemnin B brought about by the
presence of a Hiv2 group in tamandarin A. All NMR
experiments (except the variable temperature experi-
ments) were carried out in CDCl3, in which one set of
signals and therefore a single conformer of tamandarin
A (1) was observed in solution. In this solvent, all 1H and
13C chemical shifts were assigned as described earlier.
1H chemical shift values for tamandarin A (1) differed
from those of didemnin B in the same solvent by no more
than (0.23 ppm. 13C chemical shifts between the two
molecules differed up to 2.7 ppm, except in Ist1, where
C1 appeared at δ 39.9 ppm in tamandarin A (1) versus δ
29.7 ppm in didemnin B.

Tamandarins A and B: New Cytotoxic Depsipeptides
J . Org. Chem., Vol. 65, No. 3, 2000 787
a coefficient of 4.0 × 10-3 ppm/K is considered the upper
limit for internal protons that are inaccessible to solvent
and thus might be involved in hydrogen bonds.16
Ta ble 3. IC50 Cytotoxicity Va lu es in Clon ogen ic
(Colon y-F or m in g) Assa ys
tamandarin A (1)
didemnin B
(ng/mL)
(ng/mL)
The temperature coefficients (Table 4) for the amide
proton chemical shifts of tamandarin A (1) were deter-
mined in DMSO-d6. In this solvent, several conformers
of 1 can be observed. The major conformer of 1 was
assumed to be conformationally constant in solvents
ranging from CDCl3 to DMSO-d6, analogous to what has
been found for didemnin B.11 On this assumption, the
amide proton chemical shifts of the major conformer were
assigned on the basis of their coupling constants. Tem-
perature dependence was measured over the range 298-
388 K. The Ist1 and Leu3 NHs were found to be practically
temperature-independent, with very small ∆δ/∆T coef-
ficients of 9 × 10-4 and 4 × 10-5 ppm/K, respectively.
The Thr6 NH showed temperature dependence with the
larger ∆δ/∆T coefficient of 2.8 × 10-3 ppm/K. All these
coefficients, however, are less than 4.0 × 10-3 ppm/K;
hence, the amide protons of tamandarin A (1) exist in
inaccessible environments and could be involved in
hydrogen bonds. Since only a limited number of hydrogen
bonds can be formed in the molecule, these results
pointed toward a hydrogen bond situation very similar
to that of didemnin B. A weak ROE correlation between
Leu3 NH and Ist1 C2Hb, together with the small temper-
ature coefficient for Ist1, points to the involvement of Leu3
in a transannular hydrogen-bonding interaction with Ist1.
Additional ROE correlations between Ist1 C6H3 and Me2-
Tyr5 NCH3, and between Ist1 C6H3 and Pro4 CRH, are
consistent with such a hydrogen bridge across the cyclic
peptide ring. Transannular ROE correlations between
Thr6 CRH and Leu3 CâHa, and between Hiv2 CγH3 and
MeLeu7 CâHa, confirmed the internal orientation of the
Leu3 NH. These ROE correlations, consistent with the
solvent inaccessibility of the Leu3 NH, suggest orientation
of part of the linear portion of the molecule back over
the cyclic backbone and possible stabilization by hydro-
gen bonding. Strong interresidue ROE and NOE correla-
tions were observed in the linear portion of the molecule,
particularly between Pro8 CRH and Thr6 NH, MeLeu7
NCH3 and Thr6 NH, and MeLeu7 NCH3 and Pro8 CRH.
These correlations are indicative of a â II turn in the
linear side chain of the molecule, stabilized by a hydrogen
bond between Thr6 NH and Lac9 CO. The corner positions
of the turn are occupied by Pro8 in the (i + 1) position
and MeLeu7 in the (i + 2) position, bringing Pro8 CO and
MeLeu7 CRH coplanar, as can been concluded from the
low-field chemical shift, δ 5.30 ppm, of the latter.
pancreatic carcinoma
BX-PC3
prostatic cancer
DU-145
head and neck carcinoma
UMSCC10b
1.79
1.36
0.99
2.00
1.53
1.76
Ta ble 4. Tem p er a tu r e Dep en d en ce of NH P r oton s of
Ta m a n d a r in A (1)
Ist1
Leu3
Thr6
∆δ/∆T
9 × 10-4
4 × 10-5
2.8 × 10-3
Tamandarin A (1) appeared rigid, as could be deduced
from the vicinal coupling constants J NH-C4H in Ist1 (9.6
3
Hz), Leu3 (9.6 Hz), and Thr6 (5.4 Hz), which indicated a
single dominant conformation rather than freely inter-
converting residues for which average J values of 6-8
Hz would be seen. On the basis of these coupling
constants, trans conformations were assigned to the NH-
C4H and NH-CRH bonds in Ist1 and Leu3, respectively,
versus a gauche conformation for Thr6. Additional vicinal
coupling constants for Thr6, J CRH-CâH ) 1.4 Hz and
3
3J CâH-â-CH ) 6.0 Hz, and ROE enhancements between
3
Thr6 NH and Thr6 CâH and â-CH3 determined that the
conformation of this unit is practically identical to that
of Thr6 in didemnin B.11 The diastereotopic protons Ist1
C2H2 were assigned on the basis of vicinal coupling
3
3
constants J C H -C H ) 8.1 Hz and J C H -C H ) ∼0 Hz, and
2
a
3
2
b
3
their values together with the chemical shift difference
between them is another indication of the rigidity of the
molecule. For the Ist1 unit, where one could expect
significant conformational changes, additional coupling
3
3
constants of J C H
) 17.0 Hz, J C H-C H ) 9.6 Hz,
3
a-C2Hb
2
3
4
3J C H-C H ) 3.4 Hz, J C H-C H ) 6.9 Hz, and J C H-C H
)
3
4
5
6
7
5
8
6.3 Hz were observed. These values, together with NOE
enhancements between Ist1 NH and Ist1 C8H3 and C3H,
and between Ist1 C4H and Ist1 C7H3 and C5H, deter-
mined the conformation to be practically identical to that
of Ist1 in didemnin B as well.11
All remaining peptide bonds in tamandarin A (1) were
assigned trans conformations, as in didemnin B, on the
basis of NOE/ROE enhancements between NH or NCH3
and the R protons of the preceding amino acids. In the
case of Pro4 and Pro8, lacking such correlations, trans
peptide bonds were assigned on the basis of NOE
correlations between Pro4 CâH2 and Leu3 CRH and
between Pro8 CâH2 and Lac9 CRH. A trans conformation
was assigned with confidence to the bond Hiv2-Leu3, on
the basis of NOE correlations between Hiv2 CRH and
Leu3 NH. Based upon an NOE/ROE correlation between
the MeLeu7 NCH3 and Thr6 NH, a similar conclusion was
reached for the MeLeu7-Thr6 peptide bond, where NOE/
ROE correlations between MeLeu7 NCH3 and Thr6 CRH
were lacking.
The temperature dependence of amide proton chemical
shifts is generally considered to be well correlated with
their inaccessibility to solvent and hence their potential
to be involved in intramolecular hydrogen bonds. Many
studies on amide proton temperature dependence in a
variety of solvents have led to the view that ∆δ/∆T
coefficients higher than 5 × 10-3 ppm/K in polar solvents
indicate amide protons that are solvated, as in the case
of linear extended peptide conformations. Conversely,
The conformations of the side-chains in the amino acids
of tamandarin A (1) were analyzed by means of vicinal
3
coupling constants J CRH-CâH and analysis of their corre-
sponding dihedral angles ø1. As was described earlier,
based on coupling constants, the conformations of Ist1 and
Thr6 were found identical to these units in didemnin B,
which in both the crystal and solution structures adopt
dihedral angles ø1 of ∼60° and ∼155°, respectively. Hiv2,
in which one might expect the greatest changes to appear,
3
showed a vicinal coupling constant J CRH-CâH of 4.8 Hz
(versus ∼3.5 Hz in didemnin B), which indicated a gauche
conformation about the CR-Câ bond. Although the sign
of ø1 could not be unambiguously determined, the identi-
(16) (a) Pease, L. G.; Niu, C. H.; Zimmerman, G. J . Am. Chem. Soc.
1979, 101, 184-191. (b) Stevens, E. S.; Sugawara, N.; Bonora, G. M.;
Toniolo, C. J . Am. Chem. Soc. 1980, 102, 7048-7050.

788 J . Org. Chem., Vol. 65, No. 3, 2000
Ta ble 5. Obser ved NOE/ROE Cor r ela tion s for Ta m a n d a r in A (1)
Vervoort et al.
cal chemical shifts of the pro-R and pro-S methyl groups,
and thus their equal proximity to the neighboring car-
bonyl, seem to favor ø1 ) 60°, as in didemnin B. Vicinal
3
3
coupling constants J CRH-CâH and J CRH-CâH′ in Leu3 (9.6
and 1.6 Hz), Pro4 (3.6 and 7.8 Hz), Me2Tyr5 (10.8 and 4.1
Hz), and MeLeu7 (3.6 and 11.4 Hz) were extracted from
the 1D 1H NMR spectrum of tamandarin A (1). These
values are virtually identical with those found in didem-
nin B, indicating the conformations about the CR-Câ
bonds to be identical in both molecules. Therefore, the
side chain of Leu3 in tamandarin A (1) appears to be
highly restrained as in didemnin B, although the ex-
pected strong NOE correlations between Leu3 CâH and
Pro4 CγH were not observed. Coupling constants for Pro4
seemed to indicate an “endo” configuration with respect
to ring puckering, in which Cγ and CO point in the same
direction, as in didemnin B. Vicinal coupling constants
3
3J CRH-CâH and J CRH-CâH′ for Pro8, both 7.5 Hz, are quite
different from those observed in Pro4. The measured
values correspond to a dihedral angle and ring puckering
identical to Pro8 in didemnin B, which adopts an “exo”
configuration (Cγ and CO pointing in opposite directions,
Figure 3).
Tamandarin A (1) was evaluated for its cytotoxicity
(Figure 2) against three cell lines: the pancreatic carci-
noma BX-PC3 (a), the prostate carcinoma DU145 (b), and
the head and neck carcinoma UMSCC10b (c). These
bioassays were clonogenic (colony-forming) assays per-
formed under continuous exposure to tamandarin A (1)
and didemnin B. The concentrations causing a 50%
reduction in overall cell survival (IC50, Table 3) were 1.79,
1.36, and 0.99 ng/mL, respectively (versus 2.00, 1.53, and
1.76 ng/mL, respectively, for didemnin B). Although the
cell lines differed slightly in sensitivity, the two com-
pounds showed virtually the same patterns of activity
and potency.
Discu ssion
Tamandarins A and B (1 and 2) are novel depsipeptides
related to the didemnins, a well-known class of cyclic,
highly active antiviral, antitumor, and immunosuppres-
sive peptides. To date, 18 naturally occurring didemnin
congeners have been isolated from the Caribbean tuni-
cate Trididemnum solidum (class Ascidiacea, order Aplou-
sobranchia, family Didemnidae), the Mediterranean spe-
cies Trididemnum cyanophorum and Aplidium albicans
(order Aplousobranchia, family Polyclinidae), and the
F igu r e 2. Dose-response curves for tamandarin A (1) and
didemnin B, in clonogenic (colony-forming) assays, using (a)
pancreatic carcinoma BX-PC3, (b) prostatic cancer DU-145,
and (c) head and neck carcinoma UMSCC10b cell lines.
unidentified Brazilian ascidian (family Didemnidae) in
this study.3,4 Didemnin B is among the most potent

Tamandarins A and B: New Cytotoxic Depsipeptides
J . Org. Chem., Vol. 65, No. 3, 2000 789
Invariably, didemnin B was found to be toxic but inactive
in most of the trials completed. Didemnin B showed no
significant antitumor activity against these cancers, and
further clinical trials were canceled.24
Based on its structural similarity to didemnin B,
tamandarin A (1) can be expected to show potent anti-
viral, immunosuppressive, and antitumor activities; how-
ever, the former have not been confirmed. Indeed,
clonogenic cytotoxicity assays showed that cancer cell
colony formation under continuous exposure is equally
strongly inhibited by tamandarin A (1) and by didemnin
B.
The possible mechanisms through which didemnin B
may exert its antiviral, antitumor, and immunomodu-
lating activities have been studied extensively. In both
cellular (in vivo and in vitro)25 and cell-free assays,26
didemnin B was found to inhibit protein and DNA
synthesis and, to a lesser extent, RNA synthesis. Evi-
dence has been provided that inhibition of peptide
synthesis by didemnin B occurs by stabilization of
aminoacyl-tRNA binding to the ribosomal A-site, pre-
venting translocation of phenylalanyl-tRNAphe from the
A- to the P-site, but not preventing peptide bond forma-
tion.27 Consistent with these findings, it was previously
found that didemnin B binds to elongation factor 1R in a
GTP-dependent manner, and formation of the didemnin
B-GTP-EF1R complex may be responsible for the ob-
served inhibition of protein synthesis.28 The structural
similarity of tamandarin A (1) with didemnin B suggests
that they may possess the same mechanism of action.
Accordingly, in cell-free assays, tamandarin A (1) was
F igu r e 3. Computer-generated drawing of tamandarin A (1)
in solution. Most hydrogens are omitted for clarity.
members of this class of molecules, rivaled by tamandarin
A (1) in some assays. Didemnin B was early shown to be
active against several DNA and RNA viruses in vitro and
in vivo but is unfortunately ineffective against the AIDS-
causing virus HIV.3a,17,18
In several in vitro and in vivo assays, didemnin B
showed antiproliferative activity in T-lymphocytes with
a potency stronger than that of cyclosporin A, at present
the most important immunosuppressive agent used
clinicically.19 During further evaluation of the antipro-
liferative activity of didemnin B for the purpose of
prolonging organ allograft survival, it was generally
found that didemnin B displayed immunosuppressive
activity in vivo but possessed a very narrow therapeutic
index, as it displayed significant toxicity at therapeutic
dosages.20
(22) (a) Stewart, J . A.; Tong, W. P.; Hartshorn, J . N.; McCormack,
J . J . Proc. Am. Soc. Clin. Oncol. 1986, 5, 33. (b) Dorr, F. A.; Schwartz,
R.; Kuhn, J . G.; Bayne, J .; Von Hoff, D. D. Proc. Am. Soc. Clin. Oncol.
1986, 5, 39. (c) Chun, H. G.; Davies, B.; Hoth, D.; Suffness, M.;
Plowman, J .; Flora, K.; Grieshaber, C.; Leyland-J ones, B. Invest. New
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Blow, A. Cancer 1991, 68, 2550-2554. (e) Holoye, P. Y.; Raber, M. N.;
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K.; Raber, M. Cancer Chemother. Pharmacol. 1991, 29, 145-149. (g)
Dorr, F. A.; Kuhn, J . G.; Phillips, J .; Von Hoff, D. D. Eur. J . Cancer
Clin. Oncol. 1988, 24, 1699-1706. (h) Malfetano, J . H.; Blessing, J .
A.; Homesley, H. D.; Look, K. Y.; McGehee, R. Am. J . Clin. Oncol. 1996,
19, 184-186.
(23) (a) J acobs, A. J .; Blessing, J . A.; Mun˜oz, A. Gynecol. Oncol. 1992,
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Invest. New Drugs 1994, 12, 243-249. (d) Weiss, R. B.; Peterson, B.
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R.; Scher, H.; Bajorin, D.; Sternberg, C.; Bosl, G. J . Invest. New Drugs
1990, 8, 391-392. (k) Weiss, G. R.; Liu, P. Y.; O’Sullivan, J .; Alberts,
D. S.; Brown, T. D.; Neefe, J . R.; Hutchins, L. F. Gynecol. Oncol. 1992,
45, 303-306.
Didemnin B was also shown to be cytotoxic toward B16
melanoma, both in vitro and in vivo, and P388 lympho-
cytic leukemia in vivo and L1210 leukemia in vitro.21 On
the basis of these results and activity observed against
a number of human tumor stem cell lines, didemnin B
was the first marine cytotoxin to enter clinical trials as
an antineoplastic agent.22 Phase I and II clinical trials
were conducted against melanoma and cancers of the
ovaries, cervix, prostate, lung, breast, and kidney.23
(17) Rinehart, K. L., J r. In Peptides, Chemistry and Biology;
Marshall, G. R., Ed.; ESCOM: Leiden, 1988; pp 626-631.
(18) Canonico, P. G.; Pannier, W. L.; Huggins, J . W.; Rinehart, K.
L., J r. Antimicrob. Agents Chemother. 1982, 22, 696-697.
(19) (a) Teunissen, M. B. M.; Pistoor, F. H. M.; Rongen, H. A. H.;
Kapsenberg, M. L.; Bos, J . D. Transplantation 1992, 53, 875-881. (b)
Teunissen, M. B. M.; Pistoor, F. H. M.; Rongen, H. A. H.; Kapsenberg,
M. L.; Bos, J . D. J . Autoimmun. 1992, 5, XXVI. (c) Shen, G. K.; Zukoski,
C. F.; Montgomery, D. W. Int. J . Immunopharmacol. 1992, 14, 63-
74. (d) Shen, G. K.; Montgomery, D. W.; Zukoski, C. F. FASEB J . 1991,
5, A1637. (e) Montgomery, D. W.; Zukoski, C. F. Transplantation 1985,
40, 49-56.
(20) (a) Wynn, S. W.; Nelson, E. W.; Alexander, D.; Eichwald, E. J .;
Shelby, J . Clin. Res. 1990, 38, 216A (b) Yuh, D. D.; Zurcher, R. P.;
Carmichael, P. G.; Morris, R. E. Transplant. Proc. 1989, 21, 1141-
1143. (c) LeGrue, S. J .; Sheu, T.-L.; Carson, D. D.; Laidlaw, J . L.;
Sanduja, S. K. Lymphokine Res. 1988, 7, 21-30. (d) LeGrue, S. J .;
Sheu, T. L.; Sanduja, S. J . Cell. Biochem. Suppl. A 1988, 12, 226. (e)
Stevens, D. W.; J ensen, R. M.; Stevens, L. E. Transplant. Proc. 1989,
21, 1139-1140. (f) Alfrey, E. J .; Zukoski, C. F.; Montgomery, D. W.
Transplantation 1992, 54, 188-189.
(24) Goss, G.; Muldal, A.; Lohmann, R.; Taylor, M.; Lopez, P.;
Armitage, G.; Steward, W. P. Invest. New Drugs 1995, 13, 257-260.
(25) (a) SirDeshpande, B. V.; Toogood, P. L. FASEB J . 1996, 10,
A1408. (b) Crampton, S. L.; Adams, E. G.; Kuentzel, S. L.; Li, L. H.;
Badiner, G.; Bhuyan, B. K. Cancer Res. 1984, 44, 1796-1801.
(26) SirDeshpande, B. V.; Toogood, P. L. FASEB J . 1993, 7, A1083.
(27) SirDeshpande, B. V.; Toogood, P. L. Biochemistry 1995, 34,
9177-9184.
(21) J iang, T. L.; Liu, R. H.; Salmon, S. E. Cancer Chemother.
Pharmacol. 1983, 11, 1-4.
(28) Crews, C. M.; Collins, J . L.; Lane, W. S.; Snapper, M. L.;
Schreiber, S. L. J . Biol. Chem. 1994, 269, 15411-15414.

790 J . Org. Chem., Vol. 65, No. 3, 2000
Vervoort et al.
found to be a more potent inhibitor of protein biosynthesis
than didemnin B.
Although the didemnin class of cyclic depsipeptides
may not yield a clinically useful antitumor drug with
utility in conventional chemotherapy, these molecules
remain of great interest for the development of novel
cancer treatments. In view of the exceptional potency of
these cyclic peptides, they may become useful in future
applications in which antitumor compounds are delivered
directly to the tumor by means of an alternative transport
system. Such targeted systems can potentially avoid the
problem of toxicity toward normal tissues. Carrier sys-
tems that have been reported include monoclonal anti-
bodies, liposomes, viral particles, and tumor-homing
peptides.
Although the mechanism of cytotoxic action of taman-
darin A has not been thoroughly studied, it appears that
the molecule behaves similar to didemnin B. Recent
research in the laboratory of Dr. Peter Toogood at the
University of Michigan has shown that tamandarin A
inhibits protein biosynthesis in rabbit reticulocyte cell
lysates with an IC50 value of 1.3 µM. This is ap-
proximately 3 times more potent than didemnin B in the
same assay.
In view of their potent biological properties, numerous
synthetic and structure-activity studies have been per-
formed on the didemnins. The total syntheses of di-
demnins A, B, and C29 and nordidemnin B30 have been
reported, as well as the (semi)synthesis of approximately
40 “unnatural” analogues of didemnin B and its parent
structure didemnin A.31 Antitumor, antiviral, and im-
munosuppressive activities of the structures were as-
sessed in vitro and in vivo, and several compounds with
increased potency over didemnin B were discovered.
Structure-activity studies identified several parts of the
molecule as potentially important in drug-receptor
interactions. Whereas modifications of the linear side
chain resulted in increased potency in certain cases, most
stereocenters and functionalities of the cyclic depsipep-
tide core were deemed essential for bioactivity.32 It was
proposed that the keto group of the Hip2 unit plays a
major role in the bioactivities of the didemnins, since
reduction to the alcohol resulted in loss of bioactivity.3e
This proposal seems incorrect, however, since tamanda-
rin A (1), which lacks this group, shows potent cytotox-
icity and protein inhibition at levels comparable to those
observed in didemnin B.
Exp er im en ta l Section
The solution conformation of tamandarin A (1), de-
scribed in this paper, indicates that the lack of the
propionic acid unit in didemnin B results in only minor
conformational modifications. Whereas the cyclic peptide
ring in didemnin B is 23-membered, the ring in taman-
darin A (1) is 21-membered. The effect of this change
seems to be spread out over the entire molecule, resulting
in little overall change. The backbone configuration of
tamandarin A (1) appears to be stabilized by the same
three hydrogen bonds, Ist1 NH-Leu3 CO, Leu3 NH-
MeLeu7 CO, and Lac9 CO-Thr6 NH, yielding the same
“bent figure eight” shape of the cyclic peptide ring and
the side-chain folding back over the cyclic portion of the
molecule. The side-chain conformations of the amino
acids in tamandarin A (1) are almost identical to those
in didemnin B as well, indicating no major increase or
decrease in local crowding due to the reduced size of the
cyclic peptide.
Gen er a l. NMR spectra were recorded on a Varian Unity
INOVA spectrometer at 300 MHz (1H) and a Varian GEMINI
spectrometer at 100 MHz (13C) in CDCl3. FAB mass spectrom-
etry and tandem mass spectrometry were performed by K. S.
Chatman at the Scripps Research Institute, La J olla, CA.
High-performance liquid chromatography was carried out
using a reversed-phase (C18) Rainin Dynamax 60 Å column
(i.d. 10 mm) and a Waters R401 differential refractometer.
Optical rotations were determined on an Autopol III automatic
polarimeter (Rudolph Research, Flanders NJ ). UV spectra
were measured on a Perkin-Elmer Lambda 3B UV/vis spec-
trophotometer and IR spectra on a Perkin-Elmer 1600 Series
FTIR spectrophotometer.
Isola tion . Approximately 1123 g of wet ascidian was
collected in March 1996, at a depth of approximately 10 m on
a small reef off the coast of the village Tamandare´, Mamu-
cabinha, Brazil. The specimen was immediately frozen and,
upon lyophilization, yielded approximately 465 g of dry mate-
rial. The animal material was extracted three times with a
mixture of dichloromethane and methanol (1:1) to produce,
after removal of solvents in vacuo, a crude extract. The crude
extract (26.6 g) showed potent growth inhibitory activity at
or below 0.03 µg/mL in the primary in vitro cytotoxicity assay
using the HCT 116 cell line. The dark oil was partitioned
between isooctane and methanol, and the methanol fraction
was dried in vacuo and further partitioned between water and
ethyl acetate, water and dichloromethane, and water and
butanol. The ethyl acetate and dichloromethane fractions
appeared very similar by TLC analyses and contained the bulk
of the cytotoxic activity. Both solvent partitions were combined
and then fractionated by size exclusion chromatography,
employing Sephadex LH-20, using a mixture of hexane,
toluene, and methanol (3:1:1). This method has proven to be
an excellent technique for the separation of medium-sized
peptides from complex mixtures. The procedure yielded 21
fractions, all of which showed potent inhibition in the cyto-
(29) (a) Rinehart, K. L., J r.; Kishore, V.; Nagarajan, S.; Lake, R. J .;
Gloer, J . B.; Bozich, F. A.; Li, K.-M.; Maleczka, R. E. J r.; Todsen, W.
L.; Munro, M. H. G.; Sullins, D. W.; Sakai, R. J . Am. Chem. Soc. 1987,
109, 6846-6848. (b) Rinehart, K. L., J r.; Sakai, R.; Kishore, V.; Sullins,
D. W.; Li, K.-M. J . Org. Chem. 1992, 57, 3007-3013. (c) Hamada, Y.;
Kondo, Y.; Shibata, M.; Shioiri, T. J . Am. Chem. Soc. 1989, 111, 669-
673. (d) Hamada, Y.; Kondo, Y.; Shibata, M.; Shioiri, T. In Peptide
Chemistry 1987; Shiba, T., Sakakibara, S., Eds.; Protein Research
Foundation: Osaka, J apan, 1988. (e) Ewing, W. R.; Harris, B. D.; Li,
W.-R.; J oullie´, M. M. Tetrahedron Lett. 1989, 30, 3757-3760. (f) Li,
W.-R.; Ewing, W. R.; Harris, B. D.; J oullie´, M. M. J . Am. Chem. Soc.
1990, 112, 7659-7672.
(30) (a) J ouin, P.; Poncet, J .; Dufour, M.-N.; Maugrass, I.; Pantaloni,
A.; Castro, B. Tetrahedron Lett. 1988, 29, 2661-2664. (b) J ouin, P.;
Poncet, J .; Dufour, M.-N.; Pantaloni, A.; Castro, B. J . Org. Chem. 1989,
54, 617-627. (c) Ewing, W. R.; Bhat, K. L.; J oullie´, M. M. Tetrahedron
Lett. 1986, 42, 5863-5868. (d) Harris, B. D.; Bhat, K. L.; J oullie´, M.
M. Tetrahedron Lett. 1987, 28, 2837-2840.
1
(31) (a) Mayer, S. C.; Ramanjulu, J .; Vera, M. D.; Phizenmayer, A.
J .; J oullie´, M. M. J . Org. Chem. 1994, 59, 5192-5205. (b) Sakai, R.;
Rinehart, K. L.; Kishore, V.; Kundu, B.; Faircloth, G.; Gloer, J . B.;
Carney, J . R.; Namikoshi, M.; Sun, F.; Hughes, R. G., J r.; Gra´valos,
D. G.; de Quesada, T. G.; Wilson, G. R.; Heid, R. M. J . Med. Chem.
1996, 39, 2819-2834.
toxicity assay at or below 0.03 µg/mL. However, by H NMR,
one fraction appeared to be clearly enriched in peptide
metabolites. This fraction yielded 75 mg of tamandarin A (1)
accompanied by 10 mg of tamandarin B (2) after final purifica-
tion by RP (C18) HPLC using 23% water in methanol.
(32) (a) Pfizenmayer, A. J .; Ramanjulu, J .; Vera, M. D.; Ding, X.;
Xiao, D.; Chen, W.-C.; J oullie´, M. M. Bioorg. Med. Chem. Lett. 1996,
6, 2713-2716. (b) Pfizenmayer, A. J .; Ramanjulu, J .; Vera, M. D.; Ding,
X.; Xiao, D.; Chen, W.-C.; J oullie´, M. M. Abstr. Pap.-Am. Chem. Soc.
1996, 212, 172. (c) Ramanjulu, J . M.; J oullie´, M. M. Tetrahedron Lett.
1996, 37, 311-314.
Ta m a n d a r in A (1): white amorphous solid or transparent
glass; [R]D ) -35° (c ) 0.11, MeOH); IR (KBr) 3495, 3342,
2966, 2872, 1743, 1649, 1537, 1514, 1455, 1249, 1173, 1079,
1032 cm-1; UV (CH2Cl2) λmax 228 (ꢀ 24 500), 276 (ꢀ 4000), 283
nm (3400); HRFABMS (NBA/CsI matrix) m/z 1187.5074

Tamandarins A and B: New Cytotoxic Depsipeptides
J . Org. Chem., Vol. 65, No. 3, 2000 791
[MCs + 2H]+, calcd for C54H84N7O14, 1187.5131 (∆ ) 4.8 ppm);
Hz), 1.99 (m, 1H), 2.52 (d, 1H, J ) 6.0 Hz), 2.54 (s, 1H), 3.72
(s, 3H), 3.95 (m, 1H), 4.21 (m, 1H), 6.66 (br d, 1H, J ) 10.5
Hz).
1H and 13C NMR data (CDCl3) are shown in Table 1.
Ta m a n d a r in B (2): white amorphous solid or transparent
glass; [R]D ) -29° (c ) 0.11, MeOH); IR (KBr) 3472, 3342,
2966, 2872, 1742, 1661, 1637, 1531, 1514, 1449, 1249, 1173,
1078, 1032 cm-1; UV (CH2Cl2) λmax 227 (ꢀ 14 400), 277 (ꢀ 1500),
283 nm (1200); HRFABMS (NBA/CsI matrix) m/z 1173.4916
[MCs + 2H]+, calcd for C53H82N7O14, 1173.4974 (∆ ) 4.9 ppm);
1H and 13C NMR data (CDCl3) are shown in Table 2.
P r ep a r a tion of N,O-Dim eth yltyr osin e Meth yl Ester
(Me3Tyr ) Sta n d a r d . N,O-Dimethyltyrosine methyl ester was
obtained by deprotection of N-Cbz-N,O-dimethyltyrosine meth-
yl ester (1 mg, see further) in refluxing TFA (1 mL) for 30 min.
Reaction progress was monitored by silica TLC analysis. The
reaction mixture was cooled and evaporated to dryness, and
traces of TFA were removed by repeated evaporation from
water. The crude N,O-dimethyltyrosine methyl ester was then
converted to its Marfey derivative without further purification.
P r ep a r a tion of N-Cbz-N,O-Dim eth yltyr osin e Meth yl
Ester . A mixture of 100 mg (0.32 mmol) of N-Cbz-tyrosine
(D:L ) 3:7) was dimethylated using methods described previ-
ously.33 The protected amino acid was dissolved in 5 mL of
DMF, and 10 mg of Bu4NHSO4 (20 wt %) was added. Portions
of 180 mg of finely powdered KOH and 300 µL of dimethyl
sulfate were added three times over a period of 16 h, during
which time the reaction mixture was stirred vigorously at room
temperature. The reaction mixture was cooled to 0 °C and
diluted with 20 mL of diethyl ether, and the 30 mL of water
was added. The aqueous layer was separated, and the organic
layer was extracted twice with 30 mL of saturated NaHCO3
solution. The aqueous layers were combined, acidified with
KHSO4 (1 M) to pH 1, and extracted three times with ethyl
acetate. The organic layers were combined, dried over MgSO4,
filtered, and dried in vacuo. The crude reaction mixture was
dissolved in 1 mL of absolute methanol, cooled in ice, and
methylated with CH2N2 in ether. The solvent was evaporated
under a stream of nitrogen, and N-Cbz-N,O-dimethyltyrosine
methyl ester was purified by RP (C18) HPLC using 23% water
in methanol. Compound 5 was obtained as a colorless oil: 1H
NMR (CDCl3) δ 2.85 and 2.83 (s, 3H, rotational isomers), 2.97
(dd, 1H, J ) 11.9 Hz), 3.28 (tr d, 1H, J ) 14.2, 5.7 Hz), 3.75
and 3.68 (s, 3H, rotational isomers), 3.79 (s, 3H), 4.77 and 4.98
(dd, 1H, J ) 5.1, 10.2 Hz, rotational isomers), 5.05 (d, 1H, J )
5.7 Hz), 5.12 (d, 1H, 5.7 Hz), 6.78 (d, 1H, J ) 7.9 Hz), 6.82 (d,
1H, J ) 8.0 Hz), 7.04 (d, 1H, J ) 7.9 Hz), 7.13 (d, 1H, J ) 8.0
Hz), 7.24 (m, 1H), 7.33 (m, 4H).
Mild Alk a lin e Hyd r olysis of Ta m a n d a r in s A (1) a n d
B (2). A sample of tamandarin A (1) (5 mg) was dissolved in
cooled (4 °C) methanol (500 µL) and hydrolyzed by adding 30
µL of 1 N sodium hydroxide at room temperature. The
disappearance of starting material was monitored by silica
TLC. After 75 min, the reaction was quenched by acidification
with 30 µL of 1 N hydrochloric acid. The crude reaction mixture
was methylated by addition of diazomethane in ether until a
yellow coloration persisted and gas liberation ceased (ap-
proximately 1.5 mL). The resulting solution was evaporated
to dryness, and the residue was fractionated by silica column
chromatography (1 g) using 5% methanol in dichloromethane.
The two major peptide fragments produced (northern fragment
3 and southern fragment 4) were purified by RP (C18) HPLC
using 23% water in methanol. The procedure was repeated
with 5 mg of didemnin B to obtain two reference peptide
fragments (northern fragment 3 and a southern fragment).
Tamandarin B (2) (5 mg) was hydrolyzed using the same
method, yielding two peptide fragments, a northern fragment
(3′) and a southern fragment (4). Similarly, 1 mg of nordidem-
nin B was hydrolyzed to obtain reference peptide fragments
(northern fragment 3′ and a southern fragment).
Nor t h er n P ep t id e F r a gm en t of Ta m a n d a r in A (3):
white, amorphous solid or transparent glass; [R]D ) +41° (c )
0.07, MeOH); HRFABMS (NBA/CsI matrix) m/z 719.2658
[MCs + 2H+], calcd for C28H50N4O9, 719.2632 (∆ ) 3.6 ppm);
1H NMR (CDCl3, determined by 1H and COSY experiments)
for (S)-Lac, δ 4.41 (m, 1H), 1.18 (d, 3H, J ) 6.3 Hz), for L-Pro,
δ 4.75 (br tr, 1H, J ) 6.6 Hz), 3.64 (m, 2H), 2.23 (m, 2H), 1.97
(m, 2H), for D-MeLeu, δ 5.36 (dd, 1H, J ) 10.2, 5.1 Hz), 3.08
(s, 3H), 1.96 (m, 1H), 1.90 (m, 1H), 1.43 (m, 1H), 0.97 (d, 3H,
J ) 6.6 Hz), 0.93 (d, 3H, J ) 6.6 Hz), for L-Thr, δ 7.37 (d, 1H,
Acid -Ca ta lyzed Hyd r olysis of Sou th er n P ep tid e F r a g-
m en t (4). The southern peptide fragment (4, 1 mg) in 0.5 mL
of 6 N HCl was heated at 105 °C for 16 h in a sealed vial. The
cooled reaction mixture was evaporated to dryness, and traces
of HCl were removed from the residual hydrolysate by
repeated evaporation from H2O.
J
) 7.8 Hz), 4.37 (m, 2H), 1.37 (d, 3H, J ) 6.6 Hz), for
(3S,4R,5S)-Ist, δ 6.62 (br d, 1H, J ) 10.8 Hz), 4.16 (d tr, 1H,
J ) 6.0, 5.3 Hz), 4.04 (m, 1H), 3.70 (s, 3H), 2.54 (s, 1H), 2.52
(d, 1H, J ) 6.3 Hz), 1.91 (m, 1H), 1.37 (m, 1H), 1.20 (m, 1H),
0.93 (d, 3H, J ) 8.1 Hz), 0.91 (tr, 3H, J ) 6.8 Hz).
Am in o Acid An a lysis of Sou th er n P ep tid e (4) Usin g
Ma r fey’s Meth od . The previously obtained crude hydrolysate
of the southern peptide fragment (4), or the crude N,O-
dimethyltyrosine methyl ester (Me3Tyr), or a small amount
of standard free amino acid (D:L ) 3:7), in 50 µL of water/
acetone was mixed with 100 µL of a 1% solution of FDAA
(Marfey’s reagent ) 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide) in acetone. NaHCO3 (20 µL, 1 M) was added to this
mixture, and the resultant solution was heated at 40 °C for 1
h and then allowed to cool. After addition of 10 µL of 2 M HCl,
the resulting solution was evaporated, dissolved in 0.5 mL of
DMSO, and analyzed by diode array HPLC. The analysis used
the following conditions: solvent A, 0.1% TFA in H2O; solvent
B, 0.1% TFA in methanol; gradient flow rate of A + B at 1
mL/min, 100/0 to 80/20 in 45 min and from 80/20 to 40/60 in
45 min; column, Hewlett-Packard ODS Hypersil 5µ, 200 mm
× 4.6 mm; UV detection at 340 nm. The peaks were identified
by comparison with a mixture of D/L-standard amino acid-DAA
derivatives (D:L ) 3:7). Retention times (min): L-Me3Tyr-DAA,
56.5; L-Pro-DAA, 58.8; L-Leu-DAA, 70.5.
Sou th er n P ep tid e F r a gm en t of Ta m a n d a r in s A a n d
B (4): white amorphous solid or transparent glass; [R]D
)
-100°(c ) 0.03, MeOH); HRFABMS (NBA/CsI or NaI matrix)
m/z 666.2179 [MCs + H+], calcd for C28H43N3O7, 666.2155
(∆ ) 3.6 ppm), and m/z 534.3160 [M + H+], calcd for
C
28H43N3O7, 534.3179 (∆ ) 3.6 ppm); 1H NMR (CDCl3,
determined by 1H and COSY experiments) for L-Me3Tyr, δ 2.93
(s, 3H), 3.01 (dd, 1H, J ) 14.4, 9.3 Hz), 3.27, (dd, 1H, J )
14.7, 6.0 Hz), 3.80 (s, 3H), 3.69 (s, 3H), 5.03 (dd, 1H, J ) 9.3,
6.3 Hz), 6.83 (d, 2H, J ) 8.7 Hz), 7.12 (d, 2H, J ) 8.4 Hz), for
L-Pro, δ 1.87 (m, 1H), 2.16 (m, 3H), 3.68 (m, 1H), 3.82 (m, 1H),
4.78 (m, 1H), for L-Leu, δ 0.98 (tr, 6H, J ) 6.3 Hz), 1.67 (m,
1H), 1.71 (m, 2H), 4.78 (m, 1H), 6.99 (d, 1H, J ) 8.7 Hz), for
(S)-Hiv, δ 0.87 (d, 3H J ) 6.9), 1.03 (d, 3H, J ) 6.9 Hz), 2.16
(m, 1H), 3.96 (d, 1H, J ) 3.0 Hz).
Nor t h er n P ep t id e F r a gm en t of Ta m a n d a r in B (3′):
white amorphous solid or transparent glass; [R]D ) +24° (c )
0.05, MeOH); HRFABMS (NBA/NaI matrix) m/z 595.3302
[MNa + 2H+], calcd for C27H46N4O9, 595.3319 (∆ ) 2.9 ppm);
1H NMR (CDCl3, determined by 1H and COSY experiments)
for (S)-Lac, δ 1.21 (d, 3H, J ) 6.3 Hz), 4.45 (m, 1H), for L-Pro,
1.99 (m, 2H), 2.26 (m, 2H), 3.68 (m, 2H), 4.78 (br tr, 1H, J )
7.2 Hz), for D-MeLeu, δ 0.93 (d, 3H, J ) 6.6 Hz), 0.97 (d, 3H,
J ) 7.2 Hz), 1.48 (m, 1H), 1.68 (m, 1H), 1.94 (m, 1H), 3.11 (s,
3H), 5.38 (dd, 1H, J ) 10.5, 4.8 Hz), for L-Thr, δ 1.40 (d, 3H,
J ) 6.9 Hz), 4.41 (m, 2H), 7.41 (br d, 1H, J ) 7.2 Hz), for
(3S,4R)-Nst, δ 0.98 (tr, 3H, J ) 6.6 Hz), 0.99 (d, 3H, J ) 6.9
Mosh er Ester An a lysis of Hiv2 in Sou th er n P ep tid e
F r a gm en t (4). The southern peptide fragment (4, 1 mg) was
dissolved in 200 µL of dichloromethane. Dry pyridine (100 µL,
(33) The method as described in the literature (Li, W.-R.; Ewing,
W. R.; Harris, B. D.; J oullie´, M. M. J . Am. Chem. Soc. 1990, 112, 7659-
7672), followed by methylation with diazomethane, yielded only
O-methyltyrosine methyl ester. For dimethylation to occur, the modi-
fied reaction scheme was used.

792 J . Org. Chem., Vol. 65, No. 3, 2000
Vervoort et al.
predried over 4-Å molecular sieves) was added, followed by
0.5 mg of 4-(dimethylamino)pyridine. Approximately 5 µL of
(R)- or (S)-MTPA acid chloride ((R)- or (S)-R-methoxy-R-
(trifluoromethyl)phenylacetyl chloride) was added, and the
solution was left at room temperature. Reaction progress was
monitored by silica TLC analysis. After 3 days, 3 mL of
saturated NaHCO3 solution and 3 mL of diethyl ether were
added, and the solution was stirred vigorously for 30 min to
hydrolyze excess MTPA acid chloride. The organic phase was
separated, and the aqueous phase was extracted with 3 mL of
diethyl ether. The organic phases were combined, washed
three times with 3 mL of 5% aqueous NaHCO3 to remove
pyridine and three times with 3 mL of saturated NaCl solution,
dried over MgSO4, and dried in vacuo. The (R)- or (S)-MTPA
ester of southern peptide fragment (4) was purified by taking
the reaction mixture over a 1-in. silica flash column in a
Pasteur pipet using 3:1 isooctane/ethyl acetate.
ments for L-Me3Tyr, δ 2.95 (s, 3H), 3.04 (dd, 1H, J ) 14.1, 9.0
Hz), 3.27, (dd, 1H, J ) 14.1, 6.3 Hz), 3.85 (s, 3H), 3.69 (s, 3H),
5.01 (dd, 1H, J ) 9.0, 6.3 Hz), 6.81 (d, 2H, J ) 9.4 Hz), 7.12
(d, 2H, J ) 9.4 Hz), for L-Pro, δ 1.90 (m, 1H), 2.15 (m, 3H),
3.69 (m, 1H), 3.85 (m, 1H), 4.80 (m, 1H), for L-Leu, δ 0.90 (tr,
6H, J ) 6.3 Hz), 1.40 (m, 1H), 1.50 (m, 1H), 1.58 (m, 1H), 4.76
(m, 1H), 6.45 (br d, 1H, J ) 9.3 Hz), for (S)-Hiv-(R)-MTPA, δ
0.90 (d, 6H J ) 7.5 Hz), 2.28 (m, 1H), 3.58 (s, 3H), 5.16 (d, 1H,
J ) 4.5 Hz), 7.44 (m, 3H), 7.63 (d, 2H, J ) 7.5 Hz).
Ack n ow led gm en t. This research was supported by
the National Cancer Institute, NIH, under Grant
CA56770. Collaborative research in Brazil was sup-
ported by a joint NSF (INT-9403032)-CNPq grant. We
express our appreciation to Dr. Stephen B. Howell, Dr.
Gerrit Los, and David Weiner (UCSD Cancer Center)
for performing the colony-forming assays. We thank Dr.
Peter Toogood for advance information regarding pro-
tein inhibition studies with tamandarin A. We appreci-
ate the assistance of Prof. Mauro Maida and the
CEPENE group during our visit to Tamandare´.
(R)-MTP A Ester of th e Sou th er n P ep tid e F r a gm en t
(4): 1H NMR (CDCl3, determined by 1H and COSY experi-
ments) for L-Me3Tyr, δ 2.94 (s, 3H), 3.02 (dd, 1H, J ) 14.1, 9.4
Hz), 3.25, (dd, 1H, J ) 14.1, 6.5 Hz), 3.77 (s, 3H), 3.70 (s, 3H),
5.01 (dd, 1H, J ) 9.4, 6.5 Hz), 6.82 (d, 2H, J ) 9.2 Hz), 7.13
(d, 2H, J ) 9.2 Hz), for L-Pro, δ 1.90 (m, 1H), 2.15 (m, 3H),
3.70 (m, 1H), 3.77 (m, 1H), 4.80 (m, 1H), for L-Leu, δ 0.90 (tr,
6H, J ) 6.3 Hz), 1.40 (m, 1H), 1.52 (m, 1H), 1.56 (m, 1H), 4.72
(m, 1H), 6.35 (br d, 1H, J ) 9.2 Hz), for (S)-Hiv-(R)-MTPA, δ
0.97 (d, 3H J ) 7.5), 1.00 (d, 3H, J ) 7.5 Hz), 2.35 (m, 1H),
3.58 (s, 3H), 5.20 (d, 1H, J ) 3.9 Hz), 7.42 (m, 3H), 7.58 (d,
2H, J ) 6.6 Hz).
Su p p or tin g In for m a tion Ava ila ble: Spectra [NMR (1H,
13C, DEPT, COSY, TOCSY, ROESY, NOESY, g HMQC, g
HMBC), UV, IR, and MS] for tamandarins A and B. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(S)-MTP A Ester of th e Sou th er n P ep tid e F r a gm en t
(4): 1H NMR data as determined by 1H and COSY experi-
J O991425A