Onchidin B: A new cyclodepsipeptide from the mollusc Onchidium sp.

Article abstract of DOI:10.1021/ja961314i

Onchidin B (4) is a cyclic depsipeptide isolated from the pulmonate mollusc Onchidium sp. Its structure was determined by extensive 2D-NMR, FABMS, tandem FAB MS/MS, selective hydrolysis, and synthesis. It contains four α-amino acids [two units of N-methyl

Full text of DOI:10.1021/ja961314i

J. Am. Chem. Soc. 1996, 118, 11635-11643
11635
Onchidin B: A New Cyclodepsipeptide from the Mollusc
Onchidium sp.
Rogelio Ferna´ndez,‡ Jaime Rodr´ıguez,†,‡ Emilio Quin˜oa´,‡ Ricardo Riguera,*,‡
Luis Mun˜oz,§ Miryam Ferna´ndez-Sua´rez,§ and Ce´cile Debitus
Contribution from the Departamento de Qu´ımica Orga´nica, Facultad de Qu´ımica and Instituto de
Acuicultura, UniVersidad de Santiago, E-15706, Santiago de Compostela, Spain, Departamento
de Qu´ımica Pura y Aplicada, Facultad de Ciencias, UniVersidad de Vigo, E-36200, Vigo, Spain,
and Centre ORSTON, B. P. A5, Noume´a, Cedex, New Caledonia
ReceiVed April 22, 1996X
Abstract: Onchidin B (4) is a cyclic depsipeptide isolated from the pulmonate mollusc Onchidium sp. Its structure
was determined by extensive 2D-NMR, FABMS, tandem FAB MS/MS, selective hydrolysis, and synthesis. It contains
four R-amino acids [two units of N-methyl valine (MeVal), two units of proline (Pro)], four R-hydroxy acids [two
2-hydroxyisovaleric acids (Hiv), two 2-hydroxy-3-methylpentanoic acid moieties (Hmp)] and two units of the new
â-hydroxy acid: 3-hydroxy-2-methyloct-7-ynoic acid (Hymo)]. Selective hydrolysis and direct comparison by chiral
GC-MS with authentic samples of the R-amino and R-hydroxy acids allowed us the assignment of the entire absolute
stereochemistry of onchidin B. In this way, the R-hydroxy acids were found to be (S)-Hiv and (S,S)-Hmp, and the
R-amino acids (R)-proline, (S)-proline, and (R)-MeVal. In order to establish the absolute configuration of the new
â-hydroxy acid, Hymo, its four possible stereoisomers were stereoselectively synthesized using chiral N-propionyl
oxazolidinones and hex-5-ynal as starting material. Comparison by HPLC-MS of the synthetic samples with the
natural Hymo (all derivatized as esters of (-)-(R)-R-methoxy-R-(9-anthryl)acetic acid), affirmed its absolute
stereochemistry as (2R,3R). Thus, onchidin B (4) is cyclo [(R)-MeVal-(R,R)-Hymo-(S)-Pro-(S,S)-Hmp-(S)-Hiv-(R)-
MeVal-(R,R)-Hymo-(R)-Pro-(S,S)-Hmp-(S)-Hiv]. It is formed by a head-to-tail linkage of two halves, each one
built by five units and identical sequence. The lack of symmetry of onchidin B is thus due to the presence of one
(S)-Pro unit in one half and one (R)-Pro unit in the other. The structural similarity between onchidin B (4) and
onchidin (2), both isolated from the same organism, and between the â-hydroxy acid Hymo (5) and the â-amino
acid Amo (3) found in onchidin is noticed.
Introduction
isolated along the last ten years, more than a dozen dolastatins
from Dolabella auricularia.6 Particularly important from the
pharmacological point of view is the highly cytotoxic dolastatin
10, the most potent antineoplastic agent known to date. It
belongs to the selected group of marine natural products that
are in clinical or preclinical stage as antitumoral compounds.
Other marine molluscs that were reported to produce cyclic
depsipeptides are Elysia rufescens and Onchidium sp., collected
in New Caledonia, from which kahalalide F (1)7 and onchidin
(2)8 were isolated, respectively. Onchidin constitutes the only
example reported to date of a cyclic, symmetric depsipeptide
from a mollusc. Its structure is composed of two identical
halves: cyclo [(S)-N-methylvaline-(2S,3S)-3-amino-2-methyloct-
7-ynoic acid-(S)-valine-(S)-hydroxyisovaleric acid-(S)-hydroxy-
isovaleric acid]2 linked together in a head-to-tail way so onchidin
has a C2 axis of symmetry and, as a result, produces only half
the expected NMR signals. In addition to known R-amino and
R-hydroxy acids, onchidin (2) incorporates two identical units
of the new â-amino acid, 3-amino-2-methyloct-7-ynoic acid (3,
Amo).
Natural products derived from marine organisms have become
an increasingly important source of biologically active com-
pounds. Some of the most interesting ones are the cyclic
depsipeptides. This class of metabolites frequently offer an
unrivaled chemical diversity incorporating new amino and/or
hydroxy acids. From a chemical point of view, this is one of
the reasons why these compounds have attracted the effort of
researchers resulting on the report of a fair number of new
structures.1 Most of the depsipeptides from marine organisms
have been isolated from sponges; striking examples are the
geodiamolides,2 arenastatin,3 and the antifungal and cytotoxic
jasplakinolide (jaspamide).4,5
On the other hand, only three molluscs have been found to
be a source of cyclodepsipeptides. Pettit and co-workers have
† Present address: Departamento de Qu´ımica Fundamental e Industrial,
Facultad de Ciencias, Universidad de La Corun˜a, E-15071, La Corun˜a,
Spain.
‡ Universidad de Santiago.
§ Universidad de Vigo.
Centre ORSTON.
We reported some time ago several polypropionate pyrones
named onchitriols.9 Those metabolites showed moderate anti-
tumor activity and were isolated from the low polarity cytotoxic
extracts of Onchidium sp. From this marine organism, we now
X Abstract published in AdVance ACS Abstracts, October 15, 1996.
(1) Ireland, C. M; Molinski, T. F.; Roll, D. M.; Zabriskie, T. M.; MacKee,
T. C.; Swersey, J. C.; Forter, M. P. Bioorganic Marine Chemistry, Scheuer,
P. J., Ed.; Springer-Verlag, Berlin, 1989; Vol. 3, p 1.
(2) (a) Chan, W. R.; Tinto, W. F.; Manchand, P. S.; Todaro, L. J. J.
Org. Chem. 1987, 52, 3091-3093. (b) de Silva, E. D.; Andersen, R. J.;
Allen, T. M. Tetrahedron Lett. 1990, 31, 489-492. (c) Talpir, R.; Benayahu,
Y.; Kashman, Y.; Pannell, L.; Schleyer, M. Tetrahedron Lett. 1994, 35,
4453-4456.
(5) Senderowicz, A. M. J.; Kaur, G.; Sainz, E.; Laing, C.; Inman, W.
D.; Rodr´ıguez, J.; Crews, P.; Malspeis, L.; Greves, M. R.; Sausville, E. A.;
Duncan, K. L. K. J. Nat. Cancer Inst. 1995, 87, 46.
(6) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Dufresne, C.; Bates, R. B.;
Schmidt, J. M.; Cerny, R. L.; Haruhisa, K. J. Org. Chem. 1990, 55, 2989-
2990, and references cited therein.
(7) Hamann, M. T.; Scheuer, P. J. J. Am. Chem. Soc. 1993, 115, 5825-
5826.
(8) Rodr´ıguez, J.; Ferna´ndez, R.; Quin˜oa´, E.; Riguera, R.; Debitus, C.;
Bouchet, P. Tetrahedron Lett. 1994, 35, 9239-9242.
(3) Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kuroso, M.; Wang, W.;
Kitagawa, I. Tetrahedron Lett. 1994, 35, 7969-7972.
(4) (a) Crews, P.; Manes, L. V.; Boehler, M. Tetrahedron Lett. 1986,
27, 2797-2800. (b) Zabriskie, M.; Klocke, J. A.; Ireland, C. M.; Marcus,
A. H.; Molinski, T. F.; Faulkner, D. J.; Xu, C.; Clardy, J. C. J. Am. Chem.
Soc. 1986, 108, 3123-3124.
S0002-7863(96)01314-5 CCC: $12.00 © 1996 American Chemical Society

11636 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Ferna´ndez et al.
Chart 1
Table 1. 1H NMR and 13C NMR Data of Onchidin B (4) in
CDCl3
wish to report the bioassay guided isolation from the cytotoxic
CCl4 extracts (97% inhibition to Kb cells at 10 µg/mL) of
another cyclic depsipeptide, which we have named onchidin B
(4) (Chart 3), and that shares with onchidin (2) a great number
of structural features.
C no.
1H, m (J in Hz)a 13Cb
C no.
1H, m (J in Hz)a 13Cb
MeVal 1
MeVal 2
1
2
3
4
171.9 31
59.6 32
28.2 33
19.5 34
19.7 35
31.3 NMe
173.2
5.26, d (10.0)
2.15, m
0.88, d (6.5)
1.01, d (7.0)
3.21, s
5.14, d (10.0)
2.19, m
0.96, d (7.0)
1.10, d (7.0)
3.15, s
60.8
29.3
20.1
19.9
32.2
Isolation and Structural Analysis
5
Onchidium sp. (Pulmonata, order: Stylommatophora, fam-
ily: Onchidiidae) was collected at Chesterfield archipelago in
the intertidal zone on “Ile Longue” (Mouillage isles, 450 NW
of New Caledonia).
NMe
Hymo 1
Hymo 2
174.2 36
3.10, dq (2.0;7.0) 40.0 37
1.07, d (6.5) 15.0 38
5.34, dddd (10.0; 75.3 39
10.0;5.0;2.0)
6
7
8
9
175.4
39.5
14.4
3.15, m
1.13, d (7.0)
4.90, dddd (10.0; 78.2
10.0;5.0;2.0)
Kb (human epidermoid carcinoma cells) bioactivity-guided
isolation by solvent partition and further chromatography of the
methanolic extract afforded 3 mg of pure onchidin8 (2) (P-388
cells, IC50 ) 8 µg/mL) and 5 mg of onchidin B (4) [IC50 ) 8
10
11
1.79, m
1.58, m
1.46, m
30.6 40
23.5 41
1.99, m
1.67, m
1.54, m
30.8
23.2
µg/mL to Kb cells, [R]25 -220.1° (c 0.1, CHCl3)]. The (+)
D
12
2.19, dddd (16.5; 18.2 42
7.0;7.0;2.5)
2.22, dddd (16.5; 18.1
7.0;7.0;2.5)
FABMS spectrum showed a molecular ion at m/z 1153, whose
molecular formula C62H96N4O16 (17 unsaturations) was deduced
on the basis of its HRFABMS peak (m/z 1153.6999 [M + H]+,
∆ + 9.9 mmu). The edited DEPT and 13C NMR spectra showed
the presence of 62 carbons. Among them, the following signals
could be easily identified: 10 amide or ester carbonyls (δ )
175.4-166.3 ppm), 14 methyl signals (δ ) 20.1-9.4 ppm), 2
N-methyl signals (δ ) 32.2 and 31.3 ppm), 10 methines,
attached to oxygen or nitrogen (δ ) 83.5-57.6 ppm), 2
methylenes at 46.7 and 46.6 ppm, assigned as nitrogen-bearing
carbons, and another 4 signals (δ ) 83.9, 82.8, 68.9, and 68.6
ppm) that were assigned to two terminal acetylenes (Table 1).
The presence of two acetylene functional groups was confirmed
by the presence of two high field methylene carbons at δC 18.2
(C-12) and 18.1 (C-42).
13
83.9 43
82.8
68.9
14
Pro 1
15
16
17
1.91, t (2.6)
68.6 44
Pro 2
171.4 45
57.6 46
31.5 47
1.94, t (2.6)
168.9c
60.3
28.4
4.66, d (8.5)
2.12, m
4.18, d (9.0)
2.15, m
2.06, m
2.06, m
18
1.84, m
1.62, m
26.2 48
49
1.84, m
3.57, m
20.6
46.6
19
3.58, m
46.7
3.48, m
Hmp 1
20
21
22
23
Hmp 2
168.9 50
77.2 51
35.0 52
23.0 53
11.4 54
15.8 55
167.3c
83.5
34.0
25.6
9.4
5.08, d (2.5)
2.02, m
1.51, m
0.90, t (7.0)
0.98, d (7.0)
4.63, d (11.5)
1.69, m
1.22, m
0.89, t (7.0)
0.91, d (7.0)
24
25
13.6
The 1H NMR (500 MHz, CDCl3) showed as the most relevant
resonances: ten methines, corresponding to the R-amino and
R-hydroxy acid protons at δ ) 5.34 (H-9), 5.26 (H-2), 5.14
(H-32), 5.09 (H-27), 5.08 (H-21), 4.90 (H-39), 4.73 (H-57), 4.66
(H-16), 4.63 (H-51), and 4.18 (H-46) as well as two N-methyl
singlets at δ 3.21 (MeVal 1) and 3.15 (MeVal 2). In addition,
the two terminal acetylenic protons were detected as triplets
(due to propargylic coupling), at δ ) 1.94 (H-14, t, J ) 2.6
Hz) and 1.91 (H-44, t, J ) 2.6 Hz).
Hiv 1
26
27
28
29
Hiv 2
166.3 56
77.3 57
30.8 58
16.6 59
19.0 60
169.4
79.2
29.2
17.9
18.8
5.09, d (3.5)
2.38, m
0.95, d (7.0)
1.02, d (7.0)
4.73, d (8.5)
2.20, m
0.97, d (6.0)
1.03, d (6.0)
30
a Assignments based on COSY and TOCSY. b Assignments based
on HMQC and HMBC. c Assignments for these signals may be
interchanged.
The structure of 4 was determined by a detailed analysis of
one- and two-dimensional NMR spectra. A combination of
HMQC, COSY, TOCSY, and HMBC indicated the presence
of ten independent spin systems: four amino acid units and six
hydroxy acid units (Figure 1). Badly overlapped protons were
resolved by observing TOCSY correlations. The R-methine
proton of substructure 4a δ 4.66 (H-16, Pro 1) was connected
to a set of protons formed by H-17 (2.12/2.06), H-18 (1.84/
1.62), and H19 (3.58). In the same way, the signal at δ 4.18
(9) Rodr´ıguez, J.; Riguera, R.; Debitus, C. J. Org. Chem. 1992, 57, 4624-
4632.

Onchidin B
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11637
Figure 2. Selected HMBC correlations of onchidin B (4).
two spin systems belong to 2-hydroxy-3-methylpentanoic acid
units, Hmp (4d).
The remaining spin systems have the following connectivity
pattern according to COSY, HMQC, and TOCSY analysis: each
Figure 1. Partial structures 4a-e and selected COSY correlations.
1
unit possessed a methine group H/13C NMR [δ 3.10/40.0 (C-
7, Hymo 1) and 3.15/39.5 (C-37, Hymo 2)] connected to a
methyl group [δ 1.07/15.0 (C-8, Hymo 1) and 1.13/14.4 (C-38,
Hymo 2)] as well as to an oxygen-bearing methine [δ 5.34/
75.3 (C-9, Hymo 1) and 4.90/78.2 (C39, Hymo 2)] which is
bonded to a chain formed by three methylenes, the last one being
connected to a terminal acetylene [δ 1.91/68.6 (C-14, Hymo 1)
and 1.94/68.9 (C-44, Hymo 2)]. Further HMBC experiments
reaffirmed the presence of the alkyne group: correlations from
the signal at 2.19 ppm (H-12, Hymo 1) to carbons at 68.6 (C-
14, Hymo 1) and 83.9 ppm (C-13, Hymo 1) and from the proton
at 2.22 (H-42, Hymo 2) to carbons at 68.9 (C-44, Hymo 2) and
82.8 ppm (C-43, Hymo 2). This connectivity pattern is exactly
the same as we have already found in the â-amino acid Amo
(3), present in onchidin (2), but the chemical shift of the
â-carbons are notably downfield from those of Amo. All these
data led us to identify these two spin systems (4e) as belonging
to a new â-hydroxy acid, 3-hydroxy-2-methyloct-7-ynoic acid,
Hymo (5), that is reported for the first time in this work.
Relevant information pertaining to the determination of the
bonding sequence was carried out using 13C-1H long range
correlation experiments (HMBC), assigning the connectivities
from carbonyl carbons to the adjacent amino or hydroxy
substituted CR-H protons. In this way, the presence of the
structural segment Hiv 2-MeVal 1-Hymo 1 was established by
the following correlations (Figure 2): CO (C-56, Hiv 2)/H-57
(Hiv 2); CO (C-1, MeVal 1)/H-57 (Hiv 2) and H-2 (MeVal 1);
CO (C-6, Hymo 1)/H-2 (MeVal 1), NMe (MeVal 1), and Me-8
(Hymo 1). Similarly, two other skeletal segments were deduced
from the correlations shown next: CO (C-20, Hmp 1)/H-16 (Pro
1) and H21 (Hmp 1) (segment Pro 1-Hmp 1), and CO (C-31,
MeVal 2)/H-27 (Hiv 1) and H-32 (MeVal 2); CO (C-36 Hymo
2)/H-32 (MeVal 2), NMe (MeVal 2), and Me-38 (Hymo 2)
(segment Hiv 1-MeVal 2-Hymo 2).10
(H-46, Pro 2) was connected to those of H-47 (2.15/2.06), H-48
(1.84), and H-49 (3.57/3.48).
Two spin systems with N-methyl valine (MeVal), 4b, were
1
depicted as follows: H NMR resonances at δ 5.26 (Η-2, MeVal
1) showed COSY correlations to 2.15 (H-3) and this last signal
to Me-4 and Me-5 (1.01 and 0.88, respectively). The same
information was deduced from the following COSY correlation
set: 5.14 (H-32, MeVal 2)/2.19 (H-33)/1.10-0.96 (Me-34, Me-
35). One bond heteronuclear correlations allowed the identi-
fication of R-carbons, [δ 59.6 (MeVal 1, C-2) and 60.8 (MeVal
2, C-32)] and â-carbons [δ 28.2 (MeVal 1, C-3) and 29.3
(MeVal 2, C-33)] for both residues. HMBC correlations
between the N-methyl signal at 3.21 and the R-carbon at 59.6
(MeVal 1) and between the N-methyl at 3.15 and the R-carbon
at 60.8 established unambiguously the presence of the N-methyl
group in the valines.
Another two similar patterns of correlations were detected
in the COSY and TOCSY spectra for the 2-hydroxyisovaleric
acids (Hiv). Thus, the R-methine protons H-27 and H-57 [δ
5.09 (Hiv 1) and 4.73 (Hiv 2), respectively] were connected to
another two methines [δ 2.38 (H-28, Hiv 1) and 2.20 (H-58,
Hiv 2)] bonded to two pairs of methyl groups [δ 1.02/0.95 (Me-
34/Me-35, Hiv 1) and 1.03/0.97 (Me-59/Me-60, Hiv 2)].
Nevertheless, the chemical shifts of the R-carbons obtained from
HMQC [δ 77.3 (Hiv 1) and 79.2 (Hiv 2)] were significatively
downfield from those of the valines, allowing the unambiguous
assignment of the signals for the two hydroxy acid units (4c).
COSY and TOCSY for two additional spin systems showed
the following pattern of correlations: in each case, an R-methine
proton [δ 5.08 (H-21, Hmp 1) and 4.63 (H-51, Hmp 2)] was
connected to another methine [δ 2.02 (H-22, Hmp 1) and 1.69
(H-52, Hmp 2)]; this last methine was connected to both a
methyl group [δ 0.98 (H-25, Hmp 1) and 0.91 (H-55, Hmp 2)]
and a methylene group [δ 1.51 (H-23, Hmp 1) and 1.22 (H-53,
Hmp 2)]; finally, this methylene was connected to a second
methyl group [δ 0.90 (Hmp 1) and 0.89 (Hmp 2)]. HMQC
spectrum allowed the assignment of the resonances of all the
protonated carbons. The chemical shifts obtained for the
R-carbons [δ 77.2 (C-21, Hmp 1) and 83.5 (C-51, Hmp 2)] were
consistent with the presence of oxygen substituents. Thus, these
The (+) FABMS of 4 showed, in addition to [M + H]+ peak
at m/z 1153, the parent ion peak at m/z 577 (100%). HRFABMS
provided a value of m/z 577.3479 ([C31H48N2O8 + H]+, ∆ -
(10) HMBC shows also a strong correlation between C-26 (Hiv 1) and
the multiplet at δ ) 5.08-5.09 ppm. This multiplet is produced by the
overlapping of the H-21 (Hmp 1) and H-27 (Hiv 1) resonances, and so, the
connection between Hmp 1 and Hiv 1 could only be unambiguously
established after MS and selective hydrolysis.

11638 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Ferna´ndez et al.
Figure 3. Onchidin B depsipeptide sequence determination by
FABMS/MS.
0.9 mmu) that corresponds to the cleavage of the molecule into
halves with identical mass. In order to shed light on the
sequence, we decided to carry out (MS)n experiments. Thus,
collisionally induced tandem FABMS (FABMS/MS) of the m/z
577 [C31H48N2O8 + H]+ fragment gave ions at m/z 577 (Pro-
Hmp-Hiv-MeVal-Hymo + H)+, m/z 549 (Pro-Hmp-Hiv-MeVal-
Hymo - CO + H)+, m/z 480 (Hmp-Hiv-MeVal-Hymo + H)+,
m/z 464 (Hymo-Pro-Hmp-Hiv + H)+, m/z 463 (Hiv-MeVal-
Hymo-Pro + H)+, m/z 397 (Pro-Hmp-Hiv-MeVal - CO + H)+,
m/z 366 (Hiv-MeVal-Hymo + H)+, m/z 312 (Pro-Hmp-Hiv +
H)+, m/z 284 (Pro-Hmp-Hiv - CO +H)+, m/z 266 (MeVal-
Hymo + H)+, m/z 215 (Hmp-Hiv + H)+, and m/z 184 (Pro-
Hmp - CO + H)+ (Figure 3) whose assignment corroborates
the NMR results and completes the linkages between the
structural units, suggesting that onchidin B is composed by the
sequence Pro-Hmp-Hiv-MeVal-Hymo-Pro-Hmp-Hiv-MeVal-
Hymo. This accounts for only 16 unsaturation degrees and,
therefore, to satisfy the molecular formula requirements (17
unsaturations), 4 must have the corresponding cyclic structure.
Figure 4. Basic hydrolysis of onchidin B (4).
comparison with the natural one, obtained by hydrolysis of
onchidin B (4).
Diasteroselective Synthesis of Hymo
All four possible stereoisomers of 3-hydroxy-2-methyloct-
7-ynoic acid (5) were synthesized in a diastereoselective mode.
The synthetic strategy relies on the well-known reaction
developed by Evans et al.,11 between the enolborinate of
optically pure N-propionyl oxazolidinones and aldehydes to
obtain enantiomerically pure aldols and on the Mitsunobu
reaction12 which allows the inversion of configuration at the
hydroxylated carbon of the aldol, yielding its epimer.
Reaction of N-propionyl oxazolidinone 9a, derived from (S)-
phenylalaninol,13 with hex-5-ynal (prepared from hex-5-yn-1-
ol by a Swern oxidation)14 gives the aldol 10 with configuration
(2S, 3R), whereas the reaction of oxazolidinone 9b, obtained
from (1S,2R)-norephedrine, with the same aldehyde provides
aldol 11 with configuration (2R,3S).
Compounds 10 and 11 were obtained optically pure when
the reactions were carried out with freshly opened bottles of
dibutylborontriflate, since no other diastereomers were detected
by NMR. Standard hydrolysis15 of 10 and 11, followed by
diazomethane treatment, afforded â-hydroxy methyl esters 12b
(2S,3R) and 13b (2R,3S), respectively.
Stereochemistry of Onchidin B
Information about the absolute stereochemistry of onchidin
B and additional evidence of its structure was achieved by GC-
MS analysis of its components. Hydrolysis of 4 with 6 M HCl
(110 °C, 24 h) followed by derivatization and GC-MS analysis
on a chiral column (Chirasil-Val III) revealed the presence of
(S)-Hiv, (S,S)-Hmp, (R)-MeVal, (S)-Pro, and (R)-Pro in a 2:2:
2:1:1 ratio.
Furthermore, smooth basic hydrolysis (1 M NaOH/MeOH)
of 4 cleaves the ester bonds and affords, after treatment with
diazomethane, the expected three dipeptides (Figure 4): (S,S)-
Hmp-(S)-Pro-OMe (6), (S,S)-Hmp-(R)-Pro-OMe (7), and Hymo-
(R)-MeVal-OMe (8). 6 and 7 were identified by comparison
(GC-MS) with authentic samples, unambiguously confirming
the presence of both (S)-Pro and (R)-Pro in onchidin B (4). A
third component (8) from the mixture was identified by EIMS
proving the location of Hymo next to MeVal as previously
suggested by NMR.
In order to prepare the other two stereoisomers (2R,3R and
2S,3S), the inversion of one of the chiral centers of both 10
At this stage, the only remaining point to be solved was the
absolute configuration of the two asymmetric centers of the
â-hydroxy acid Hymo. No useful J values could be obtained
due to massive overlapping in the 1H NMR spectrum. A strong
NOE was detected between H-7/H-9 and H-37/H-39, but it was
not conclusive to distinguish between eritro or threo relative
stereochemistries. To overcome this difficulty, we decided to
prepare the four stereoisomers of Hymo and proceed to their
(11) Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc. 1981, 103,
2127-2129.
(12) (a) Mitsunobu, O. Synthesis 1981, 1-28. (b) Hugues, D. L. Org.
React. 1992, 42, 335-656.
(13) Gage, J. R.; Evans, D. A. Org. Synth. 1989, 68, 77-91.
(14) Freeman, F.; Kim, D. S. H. L.; Rodr´ıguez, E. J. Org. Chem. 1992,
57, 1722-1727.
(15) Evans, D. A.; Britton, T. C.; Ellman, J. A. Tetrahedron Lett. 1987,
28, 6141-6144.

Onchidin B
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11639
Scheme 1a
a (a) (nBu)2BOTf, Et3N, CH2Cl2, 0 °C and then hex-5-ynal, CH2Cl2, -70 °C; (b) LiOH, H2O2, THF-H2O, 0 °C; (c) CH2N2, Et2O; (d)
diethylazodicarboxylate, triphenylphosphine, p-nitrobenzoic acid, benzene; (e) 30% H2O2, LiOH, 0 °C.
Chart 2
and 11 was needed. Since the configuration at position 2 could
not be fully inverted easily, we decided to concentrate our efforts
on the inversion of the configuration at carbon 3 making use of
the Mitsunobu reaction. When â-hydroxy methyl ester 12b was
submitted to Mitsunobu conditions, no successful results were
obtained. Nevertheless, a smooth inversion took place when
aldols 10 and 11 were treated in similar conditions16 and
p-nitrobenzoate esters 14 and 15 were obtained in good yields.
Simultaneous hydrolysis of the benzoate ester and oxazolidinone
moieties, followed by diazomethane treatment, afforded the
desired (2S,3S)-â-hydroxy methyl ester 16 and its (2R,3R)
isomer 17, in good yields.
Direct comparison of the methyl esters of the four synthetic
stereoisomers with the methyl ester of Hymo, the natural
component of onchidin B (4), by chiral GC and HPLC was not
successful in spite of our efforts to find experimental conditions
suitable for a good separation of the four isomers. This problem
was finally overcome by HPLC-MS analysis of the 3-O-[(-)-
(R)-R-methoxy-R-(9-anthryl)acetyl]-2-methyloct-7-ynoic methyl
esters (18-21). These derivatives were prepared by standard
treatment of the hydroxy esters with (-)-(R)-R-methoxy-R-(9-
anthryl)acetic acid,17 and were chosen because they provide four
beautifully separated peaks in the HPLC chromatogram. In
addition, they present strong and characteristic UV absorption
and give clean CIMS fragmentations that enabled a safe
comparison by coinjection with the natural Hymo.
Thus, reaction of the four synthetic â-hydroxy esters with
(-)-(R)-R-methoxy-R-(9-anthryl)acetic acid, in the presence of
DCC and DMAP, afforded the corresponding derivatives that
showed the following retention times in HPLC-MS with the
mass detector fixed at m/z 378: (S,R) 28.09 min, (R,R) 30.93
min, (R,S) 41.74 min, and (S,S) 46.54 min.
Onchidin B (4) was hydrolyzed with 6 M HCl at 110 °C for
24 h, and the mixture containing the free Hymo was esterified
first with diazomethane and then with (-)-(R)-R-methoxy-R-
(9-anthryl)acetic acid. HPLC-MS of that mixture showed a peak
with identical tR, UV, and MS as isomer (2R,3R)-3-O-[(-)-
(R)-R-methoxy-R-(9-anthryl)acetyl]-2-methyloct-7-ynoic methyl
ester. Therefore, the absolute configuration of the natural Hymo
(5) is (2R,3R) and the structure of onchidin B (4) is cyclo [(R)-
MeVal-(R,R)-Hymo-(S)-Pro-(S,S)-Hmp-(S)-Hiv-(R)-MeVal-(R,R)-
Hymo-(R)-Pro-(S,S)-Hmp-(S)-Hiv].
Finally, in order to assign the (R) and (S) stereochemistries
to the prolines, characteristic 13C chemical shifts differences18
and additional NOEs were considered. The linkage between
Pro 1 and Hmp 1 is probably trans, as suggested by the value
obtained for ∆δâγ ) 5.3 ppm for C-17 and C-18, while the
bond between Pro 2 and Hmp 2 is probably cis in accordance
with the value ∆δâγ ) 7.8 ppm for C-47 and C-48. A ROESY
correlation between H-21 (Hmp 1) and H-19 (Pro 1) confirms
the trans assignment for Pro 1.19 These considerations, together
with the NOE observed between H-21 (Hmp 1) and H-16 (Pro
1),20 suggest that both hydrogens lie on the same side, so C-16
(16) Sone, H.; Kondo, T.; Kiryu, M.; Ishiwata, H.; Ojika, M.; Yamada,
K. J. Org. Chem. 1995, 60, 4774-4781.
(17) Latypov, Sh. K.; Seco, J. M.; Quin˜oa´, E.; Riguera, R. J. Org. Chem.
1995, 60, 504-515.
(18) Siemion, I. Z.; Wieland, T.; Pook, K.-H. Angew. Chem., Int. Ed.
Engl. 1975, 14, 702-703.

11640 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Ferna´ndez et al.
increment. FABMS were obtained on a Kratos MS-50 (employing Xe
atoms at 7-9 KeV; glycerol and 2-hydroxyethyl disulfide + NaCl
matrix) and FINIGAN 4000 (NBA matrix). Collisionally induced
tandem mass spectra (MS/MS) in the FAB mode were obtained on a
four sector tandem mass spectrometer VG 70-SE-4F. EIMS was
obtained on a Hewlett-Packard HP59970 spectrometer.
Chart 3
HPLC separations were performed on a Waters Model 6000A using
reversed-phase µ-Bondapack C18 (7.8 mm I.D. × 30 cm) and µ-porasil
(7.8 mm I.D. × 30 cm). Gas chromatography analyses were performed
using a Hewlett-Packard spectrometer with a Chirasil-Val III capillary
column (50 m × 0.25 mm).
Cytotoxic activity was assessed in Vitro using Kb cells.24
(1S,2R)-Norephedrine, (S)-phenylalanine, hex-5-yn-1-ol, and dibu-
tylboron triflate were purchased from Aldrich Chemical Company and
were used without further purification. All solvents were distilled under
argon utilizing standard literature procedures. Flash column chroma-
tography was performed using Kieselgel 60 230-400 mesh SiO2 gel
into glass columns. Synthetic products were purified by HPLC before
optical rotation measurements. Synthetic yields were not optimized.
Extraction and Isolation. Animal Collection. Onchidium sp. is
a Pulmonata, order: Stylommatophora, family: Onchidiidae, collected
in July 1988 from Chesterfield archipelago in the intertidal zone on
“Ile Longue” (Mouillage isles, 450 NW of New Caledonia), sample
reference MG 332. Each animal weight was 300 g approximately. A
voucher specimen is in deposit at the Museum National d′Histoire
Naturelle in Paris, and it has been identified by Dr. Philippe Bouchet.
Extraction and Partitioning. Three kilograms of mollusc were
freeze-dried (600 g dry weight) and then extracted with methanol (4
× 2 L). The methanol extract was decanted off and concentrated in
vacuum. The viscous concentrate was partitioned between 400 mL of
10% aqueous methanol and hexanes (2 × 400 mL). The methanolic
portion was made 20% aqueous and extracted with CCl4 (2 × 400 mL).
Then the methanolic portion was made 40% aqueous and extracted
with CH2Cl2 (3 × 400 mL). The organic layers were concentrated in
vacuum to yield 2.9 g of hexane extract (15% inhibition to Kb cells at
10 µg/mL), 1.2 g of CCl4 extract (97% inhibition to Kb cells at 10
µg/mL), and 0.7 g of CH2Cl2 extract (99% inhibition to Kb cells at 10
µg/mL).
and C-21 must have identical configuration and Pro 1 should
be (S). Furthermore, the lack of NOE between H-46 (Pro 2)
and H-51 (Hmp 2), in spite of the cis stereochemistry, suggests
that both protons do not lie on the same side of the molecule,
and so the absolute configuration of Pro 2 should be (R).
Onchidin B (4) is then a cyclic depsipeptide formed by the
head-to-tail bonding of two chains (MeVal-Hymo-Pro-Hmp-
Hiv) with identical sequence. In spite of this common sequence,
onchidin B (4) has no C2 symmetry and shows signals for all
the 62 carbons in the NMR spectra. This lack of coincidence
of NMR signals is related to the presence of the two enantiomers
of proline that renders the two halves of the molecule different.
Similar structural characteristics were found in onchidin (2),
the other cyclodepsipeptide isolated from this organism, but,
in that case, the repeating amino and hydroxy acids have the
same configuration and so compound (2) has C2 symmetry and
presents only half the expected proton and carbon resonances.
A further point of interest is the presence in onchidin B (4)
and onchidin (2) of the obviously related acetylenic â-hydroxy
and â-amino acids, [Hymo (5) and Amo (3)], respectively. A
â-hydroxy acid with the same skeleton but an additional methyl
group at C-2 has recently been described by P. Scheuer as a
component of the cyclodepsipeptide kulolide isolated from the
mollusc Philinopsis speciosa.21 Another related molecule is the
â-hydroxy acid, (2R,3S)-7,7-dichloro-3-hydroxy-2-methyloc-
tanoic acid, isolated from the marine sea hare Dolabella
auricularia16 that can be viewed as the dichlorinated derivative
of a Hymo stereoisomer.
The CCl4 extract was subjected to flash silica gel column chroma-
tography (3 × 20 cm, silica gel 60, Merck 70-230 mesh) stepped
gradient elution from 100% dichloromethane to 100% methanol giving
five fractions: F1 (inactive), F2 (cytotoxic to Kb cells at 10 µg/mL:
93% inhibition), F3 (inactive), F4 (cytotoxic to Kb cells at 10 µg/mL:
95% inhibition), and F5 (inactive).
Isolation of Onchidin B. Fraction F2 was purified on a reversed-
phase µ-Bondapack C18 HPLC column (30 cm × 7.8 mm, 93:7 MeOH:
H2O; flow rate: 2.0 mL/min) affording 5 mg (17 × 10-2 % from the
D
CCl4 extract) of onchidin B (4), tR 12.7 min; [R]25 -220.1° (c 0.1,
Experimental Section
CHCl3); FTIR (KBr) 3280, 2980, 2940, 2880, 1747, 1650, 1640 cm-1
;
HRFABMS calcd for C62H97N4O16 m/z 1153.6899 [M + H]+, found
1153.6999, ∆ + 9.9 mmu; HRFABMS calcd for C31H49N2O8 m/z
577.3488 [M + H]+, found 577.3479, ∆ - 0.9 mmu; (+) FABMS
(NBA matrix): m/z (% relative intensity): 1153 [M + H]+ (7), 577
(100). (+) FABMS (2-hydroxyethyl disulfide + NaCl matrix), m/z
(% relative intensity): 1175 [M + Na]+ (1), 1153 [M + H]+ (5), 577
(100). 1H and 13C NMR see Table 1.
General Methods. IR and UV spectra were obtained on MIDAC
Prospect FTIR and 8452 A spectrophotometers, respectively, and optical
rotations on a Jasco DIP-370 digital polarimeter. NMR spectra were
recorded on Bruker AMX 500, Bruker AMX 300, or Bruker WM 250
spectrometers using CDCl3 as solvent and internal standard. The 1H-
1H COSY spectrum was acquired with 4K × 354 matrix and eight
scans per increment. The TOCSY spectrum was acquired with 4K ×
300 matrix and 16 scans per increment. The ROESY spectrum was
acquired with 2K × 200 matrix and 32 scans per increment. The
TOCSY and ROESY experiments were recorded using a mixing time
of 0.070 and 0.280 ms, respectively. The HMQC22 and HMBC23
sequences were performed with 2K × 200 matrix and 64 scans per
(19) Masashi, T.; Shigemori, H.; Mikami, Y.; Kobayashi, J. Tetrahedron
1993, 49, 6785-6796.
(20) According to the literature, NOEs between H-16 and H-21 should
be expected if the linkage Pro 1-Hmp 1 were cis and between H-19 and
H-21 if trans. Models indicate that in 4 both NOEs are possible depending
on the flexibility of the molecule and, in fact, we have observed them
experimentally.
Amino Acid Analysis by Chiral GC-MS. Onchidin B (4) (1 mg)
was submitted to hydrolysis with 6 M HCl (0.5 mL) in a sealed tube
at 110 °C for 24 h. The excess HCl was removed by passing a stream
of N2, and the residue was dried under vacuum. The hydrolyzates were
diluted with water (1 mL), and the hydroxy acids were extracted with
diethyl ether (3 × 1 mL).
The ethereal solution was dried under vacuum, and the residue was
dissolved in 1 mL of ether and treated with diazomethane for 30 min.
The excess of reagent was removed with a stream of dry N2. Capillary
(23) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093-
2094.
(21) Nakao, Y.; Juagdan, E. G.; Yoshida, W. Y.; Scheuer, P. J. Eighth
International Symposium On Marine Natural Products; Santa Cruz de
Tenerife, Canary Islands, September 10-15, 1995.
(22) Bax, A.; Subramanian, S. J. Am. Chem. Soc. 1985, 107, 2820-
2821.
(24) Bergeron, R. J.; Cavanaugh, P. F., Jr.; Kline, S. J.; Hughes, R. G.,
Jr.; Elliot, G. T.; Porter, C. W. Biochem. Biophys. Res. Comm. 1984, 121
(3), 848-854.
(25) McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51, 1915-
1919.

Onchidin B
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11641
GC-MS analysis were carried out using a Chirasil-Val III column (50
m × 0.25 mm; carrier gas: He; flow rate: 1 mL/min; column
temperature: 50 °C, 60 min isothermal) to show peaks at tR 15.39 and
33.50 min. Standards (S)- and (R)-Hiv and (S,S)-, (R,S)-, (R,R)- and
(S,R)-Hmp were also converted to the methyl derivatives by the same
procedure. Retention times (minutes) were as follows: 15.51 [(S)-
Hiv], 16.18 [(R)-Hiv], 31.10 [(S,R)-Hmp], 32.50 [(R,S)-Hmp], 33.69
[(S,S)-Hmp], and 35.25 [(R,R)-Hmp].
The water solution was dried under vacuum, and the resulting residue
was divided into two equal portions. One of them was dissolved in
dichloromethane (0.5 mL) and N-isopropyl isocyanate (1 mL) was
added in a screwed-cap vial. After 60 min at 100 °C the excess of
reagent was removed with a steady stream of dry N2. Capillary GC-
MS analysis were carried out using a Chirasil-Val III column (50 m ×
0.25 mm; carrier gas: He; flow rate: 1 mL/min; column temperature
150 °C, 45 min isothermal, then programmed an increase of 5 °C/min
to 200 °C) to show peak at tR 33.11 min. Standards (S)- and (R)-
MeVal [available by methylation of (S)- and (R)-Val]25 were also
converted to the N-isopropyl isocyanates by the same procedure.
Retention times (min) were as follows: 31.02 and 33.30 for (S)- and
(R)-N-methyl valine, respectively.
The second portion was dissolved in n-BuOH (0.5 mL) and HCl (3
M, 0.5 mL) and heated in a sealed tube at 100 °C for 30 min. The
product was evaporated, dissolved in trifluoracetic anhydride (0.5 mL)
and CH2Cl2 (0.5 mL), reacted in a sealed tube at 150 °C for 10 min
and evaporated in a stream of nitrogen. Capillary GC-MS analysis
were carried out using the same column Chirasil-Val III column (50
m × 0.25 mm; carrier gas: He; flow rate: 1 mL/min; column
temperature 50 °C, 30 min isothermal then programmed an increase
of 2 °C/min to 200 °C) to show peaks at tR 60.24 and 60.42 min.
Standard (S)- and (R)-Pro were also derivatized by the same procedure.
Retention times (min) were as follows: 60.36 and 60.55 for (R)- and
(S)-proline, respectively.
Alkaline Hydrolysis of Onchidin B. Onchidin B (4) (0.5 mg) in
MeOH (0.5 mL) was treated under an argon atmosphere with NaOH
(1 M, 0.5 mL) at 0 °C. After 1 h the reaction mixture was diluted
with CH2Cl2 (2 mL) and water (1 mL). The aqueous phase was
acidified to pH 2 with 1 M HCl and extracted with CH2Cl2 (2 × 2
mL). The CH2Cl2 extract was dried with anhydrous Na2SO4 and
concentrated in vacuum. The residue dissolved in ether (1 mL) was
treated with an excess CH2N2 at room temperature for 30 min, and the
excess of reagent was removed with a steady stream of dry N2.
Capillary GC-MS analysis were carried out using a Chirasil-Val III
column (50 m × 0.25 mm; He as carrier gas; flow rate 1 mL/min;
column temperature 150 °C, 45 min isothermal then programmed at 5
°C/min to 200 °C) to show peaks at tR 62.86 min [(S,S)-Hmp-(R)-Pro-
OMe EIMS m/z (% relative intensity): 211 (6), 155 (74), 127 (23)];
63.97 min [(S,S)-Hmp-(S)-Pro-OMe EIMS m/z (% relative intensity):
211 (1), 155 (99), 127 (30)] and 69.37 min [(Hymo-MeVal-OMe EIMS
m/z (% relative intensity): 267 (1), 238 (20), 230 (20), 201 (4), 144
(19), 86 (100)].
2), 296 (10), 244 (20), 233 (42), 196 (30), 178 (46), 153 (14), 148
(17), 134 (82), 91 (100), 85 (71), 57 (25). HREIMS: calcd for
C19H23O4N m/z 329.1627 (M+), found 329.1629. FTIR (CHCl3): 3526,
3289, 2940, 2872, 1777, 1691, 1453, 1386, 1213, 1107, 972 cm-1. 1H
NMR (250.13 MHz, CDCl3): δ 1.22 (d, J ) 7.1 Hz, 3 H), 1.47-1.75
(m, 4 H), 1.90 (t, J ) 2.3 Hz, 1 H), 2.17-2.25 (m, 2 H), 2.74 (dd, J
) 13.3, 9.4 Hz, 1 H), 2.91 (s broad, 1 H), 3.20 (dd, J ) 13.3, 3.3 Hz,
1 H), 3.70 (m, 1 H), 3.93 (m, 1 H), 4.11-4.23 (m, 2 H), 4.65 (m, 1
H), 7.14-7.35 (m, 5 H). 13C NMR (62.89 MHz, CDCl3): δ 10.3 (c),
18.1 (t), 24.8 (t), 32.6 (t), 37.7 (t), 42.1 (d), 55.0 (d), 66.1 (t), 68.5 (s),
70.9 (d), 84.1 (d), 127.4 (d), 129.0 (d), 129.4 (d), 135.0 (s), 153.0 (s),
177.5 (s).
(4R,5S,2′R,3′S)-3-(3′-Hydroxy-2′-methyl-7′-octynoyl)-5-phenyl-4-
methyl-2-oxazolidinone (11). Using the method described for the
preparation of (4S,2′S,3′R)-3-(3′-hydroxy-2′-methyl-7′-octynoyl)-4-(phe-
nylmethyl)-2-oxazolidinone (10), N-propionyl oxazolidinone 9b (3.875
g, 16.63 mmol) was treated with 1 M dibutylborontriflate (18.3 mL,
18.3 mmol) and triethylamine (3 mL, 21.62 mmol) and the resulting
enolborinate was allowed to react with hex-5-ynal (2.1 g). After workup
and purification the aldol 11 was obtained as a white solid (mp 97-
25
100 °C), (4.1 g, 75% yield). [R]D +8.3 (c 1.6, CHCl3). EIMS m/z
(% relative intensity): 262 (M+, 9), 234 (15), 233 (100), 178 (20),
134 (36), 188 (42), 107 (60), 91 (21), 57 (25). HREIMS: calcd for
C19H23NO4 m/z 329.1627 (M+) found 329.1630. FTIR (CHCl3): 3521,
3292, 2940, 2875, 1779, 1694, 1455, 1351, 1231, 1197, 1120, 960 cm-1
.
1H NMR (300.13 MHz, CDCl3): δ 0.89 (d, J ) 6.6 Hz, 3 H), 1.25 (d,
J ) 6.9 Hz, 3 H), 1.54-1.79 (m, 4 H), 1.96 (t, J ) 2.3 Hz, 1 H),
2.22-2.27 (m, 2 H), 2.78 (s broad, 1 H), 3.78 (m, 1 H), 4.00 (m, 1 H),
4.80 (m, 1 H), 5.69 (d, J ) 7.3 Hz, 1 H), 7.26-7.46 (m, 5 H). 13C
NMR (75.45 MHz, CDCl3): δ 10.6 (c), 14.7 (c), 18.6 (t), 25.3 (t),
33.1 (t), 42.6 (d), 55.1 (d), 69.0 (s), 71.4 (d), 79.3 (d), 84.5 (d), 126.0
(d), 129.1 (d), 129.2 (d), 133.4 (s), 153.0 (s), 177.7 (s).
Methyl (2S,3R)-3-Hydroxy-2-methyl-7-octynoate (12b). A solu-
tion of the aldol 10 (1.24 g, 3.76 mmol) in THF:H2O (4:1, 20 mL) was
cooled to 0 °C. To this solution were sequentially added 30% H2O2
(1.53 mL, 15.04 mmol) and a solution of LiOH (252 mg, 6.0 mmol) in
water (5 mL). The solution was stirred for 1 h. A solution of Na2SO3
(1.89 g, 15.04 mmol) in water (12 mL) was added, and the resulting
solution was stirred for another 15 min. The organic solvent was
concentrated under reduced pressure, and the resulting aqueous phase
(pH 12) was extracted with dichloromethane (3 × 40 mL). Then the
aqueous phase was cooled to 0 °C, acidified to pH 1 with 1.5 M HCl,
and extracted with EtOAc (5 × 40 mL). The organic phase was dried
over anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The â-hydroxy acid 12a was obtained as a viscous colorless
oil (610 mg, 95% yield). The dichloromethane extract was dried over
MgSO4 and filtered, and the solvent was removed under reduced
pressure. In this way the chiral oxazolidinone auxiliary was recovered
25
as a white solid (565 mg, 85% yield). [R]D +6.2 (c 2.0, CHCl3).
EIMS m/z (% relative intensity): 170 (M+, 1), 137 (4), 124 (4), 103
(35), 85 (43), 74 (100), 57 (21). HREIMS: calcd for C9H14O3 m/z
170.0942 (M+) found 170.0937. FTIR (CHCl3) 3453, 3292, 2943,
(4S,2′S,3′R)-3-(3′-Hydroxy-2′-methyl-7′-octynoyl)-4-(phenylmethyl)-
2-oxazolidinone (10). A stirred solution of N-propionyloxazolidinone
9a (3.0 g, 12.9 mmol) in dry dichloromethane (30 mL) was cooled to
0 °C and sequentially treated dropwise via syringe with 1 M dibutyl-
boron triflate (18.0 mL, 18.0 mmol in dichloromethane) and triethyl-
amine (3.0 mL, 2.2 g, 21.8 mmol). The resulting solution was cooled
to -70 °C, and a solution of hex-5-ynal (1.9 g) in dichloromethane
(10 mL) was added dropwise via syringe. The resulting mixture was
stirred at -60 °C for 20 min and then an additional hour at 0 °C. The
reaction was quenched by addition of aqueous phosphate buffered
solution (pH 7, 15 mL) and methanol (15 mL). Then a mixture
methanol:30% H2O2 (2:1, 20 mL) was added, and the resulting solution
was stirred for 45 min at room temperature. The solvent was evaporated
under reduced pressure, the residue was redissolved in water (40 mL),
and the resulting solution was extracted with EtOAc (3 × 20 mL).
The combined organic phase was washed with 5% NaHCO3 (40 mL)
and brine (40 mL), dried over anhydrous MgSO4, and concentrated
under reduced pressure, yielding a viscous yellow oil. The crude was
purified by flash chromatography (EtOAc:hexanes 1:3) and the aldol
1709, 1456, 1408, 1210, 1078, 958 cm-1 1H NMR (300.13 MHz,
.
CDCl3): δ 1.20 (d, J ) 7.2 Hz, 3 H), 1.52-1.79 (m, 4 H), 1.96 (t, J
) 2.6 Hz, 1 H), 2.21-2.26 (m, 2 H), 2.59 (dd, J ) 7.2, 3.3 Hz, 1 H),
3.98 (m, 1 H), 6.51 (s broad, 2 H). 13C NMR (75.47 MHz, CDCl3):
δ 10.7 (c), 18.6 (t), 25.2 (t), 32.9 (t), 44.6 (d), 69.1 (d), 71.6 (s), 84.4
(d), 181.4 (s).
N-Nitroso-N-methyl urea (150 mg) was added to a two-phase 50%
aqueous KOH:ether system (30 mL, 1:2). When the gas evolution
ceased, the organic phase was carefully decanted and added to a solution
of the â-hydroxy acid 12a (57 mg, 0.33 mmol) in ether (10 mL) at
room temperature. After 30 min the solvent was evaporated with an
argon flow, yielding the â-hydroxy methyl ester 12b as a yellowish
25
oil. [R]D +14.3 (c 0.4, CHCl3). (+) FABMS (glycerol matrix) m/z
(% relative intensity): 185 [M + H]+ (89), 167 (15), 153 (35), 125
(11), 107 (78). HREIMS: calcd for C10H16O3 m/z 184.1099 (M+),
found 184.1099. FTIR (CHCl3) 3507-3465, 3294, 2946, 1728, 1448,
1434, 1368, 1264, 1201, 1167, 1087, 1046 cm-1 1H NMR (300.13
.
MHz, CDCl3): δ 1.19 (d, J ) 7.2 Hz, 3 H), 1.51-1.64 (m, 3 H), 1.70-
1.80 (m, 1 H), 1.95 (t, J ) 2.6 Hz, 1H), 2.23 (m, 2 H), 2.51-2.59 (dq,
J ) 7.2, 3.5, 1 H), 3.71 (s, 3 H), 3.91 (m, 1 H). 13C NMR (62.89
25
10 was obtained as a colorless viscous oil (3.4 g, 80% yield). [R]D
+29.3° (c 1.2, CHCl3). EIMS m/z (% relative intensity): 329 (M+,

11642 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Ferna´ndez et al.
MHz, CDCl3): δ 10.6 (c), 18.1 (t), 24.8 (t), 32.7 (t), 44.3 (d), 51.7 (c),
68.6 (d), 71.3 (d), 84.1 (s), 176.5 (s).
H2O (40 mL, 4:1) was sequentially treated with 30% H2O2 (0.43 mL,
3.78 mmol) and a solution of LiOH (120 mg, 2.86 mmol) in H2O (1
mL). The mixture was stirred for 1 h at 0 °C and then for a period of
18 h at room temperature. Aqueous 10% Na2SO3 solution (5 mL) was
added, and the organic solvent was removed under reduced pressure.
The resulting aqueous phase was extracted with dichloromethane (4 ×
30 mL), acidified to pH 1 with 6 M HCl, and reextracted with EtOAc
(4 × 30 mL). The combined EtOAc extract was washed with brine
(50 mL), dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was redissolved in ether (15 mL) and
was treated with an ethereal solution of diazomethane. The solvent
was evaporated with an argon flow, and the crude was purified by flash
chromatography (EtOAc:hexanes 1:8), yielding the â-hydroxy methyl
ester 16 as a colorless viscous oil (120 mg, 76%). [R]D25 +2.5 (c 0.5,
CHCl3). (+) FABMS (glycerol matrix) m/z (% relative intensity): 185
[M + H]+ (100), 167 (6), 153 (30), 125 (13), 107 (59). EIMS m/z (%
relative intensity): 173 (1), 151 (2), 143 (2), 117 (56), 107 (7), 97 (9),
88 (100). HREIMS calcd for C10H16O3 m/z 184.1099 (M+), found
184.1099. 1H NMR (300.13 MHz, CDCl3): δ 1.21 (d, J ) 7.2 Hz, 3
H), 1.45-1.83 (m, 4 H), 1.94 (t, J ) 2.6, 1 H), 2.22 (m, 2 H), 2.52 (q,
Methyl (2R,3S)-3-Hydroxy-2-methyl-7-octynoate (13b). Aldol 11
was hydrolyzed using the method described for the hydrolysis of 10.
Thus, aldol 11 (888 mg, 2.7 mmol) was treated with 30% H2O2 (1.1
mL, 10.84 mmol) and LiOH (182 mg, 4.3 mmol). After workup and
purification the chiral auxiliary oxazolidinone was recovered (425 mg,
88% yield), and the â-hydroxy acid 13a was obtained as a colorless
25
viscous oil (440 mg, 95% yield). [R]D -6.0 (c 1.3, CHCl3). NMR
and MS spectroscopic data are identical to those obtained for 12a.
13b was obtained by treatment of â-hydroxy acid 13a with excess
diazomethane as described previously for â-hydroxy methyl ester 12b.
25
NMR spectroscopic data are identical to those obtained for 12b. [R]D
-13.0 (c 0.7, CHCl3). (+) FABMS (glycerol matrix) m/z (% relative
intensity): 185 [M + H]+ (93), 167 (23), 153 (41), 125 (18), 107 (71).
HREIMS: calcd for C10H16O3 m/z 184.1099 (M+), found 184.1099.
1H NMR (300.13 MHz, CDCl3): δ 1.19 (d, J ) 7.3 Hz, 3 H), 1.51-
1.61 (m, 2 H), 1.75 (m, 1 H), 1.95 (t, J ) 2.6 Hz, 1 H), 2.23 (m, 2 H),
2.53-2.57 (m, 2 H), 3.71 (s, OMe, 3 H), 3.91 (m, 1 H). 13C NMR
(62.89 MHz, CDCl3): δ 10.6 (c), 18.1 (t), 24.8 (t), 32.6 (t), 44.3 (d),
51.7 (c), 68.5 (d), 71.2 (d), 84.1 (s), 176.5 (s).
J ) 7.1, 1 H), 2.66 (d, J ) 6.4, 1 H), 3.67 (m, 1 H), 3.70 (s, 3 H). 13
C
NMR (75.47 MHz, CDCl3): δ 14.7 (c), 18.6 (t), 24.8 (t), 33.9 (t), 45.6
(d), 52.2 (c), 69.0 (d), 73.2 (d), 84.6 (s), 176.8 (s).
(4S,2′S,3′S)-3-[2′-Methyl-3′-[(4′′-nitrobenzoyl)oxy]-7’-octynoyl]-
4-phenylmethyl-2-oxazolidinone (14). A solution of diethylazodi-
carboxylate (1.48 g, 8.47 mmol) in benzene (5 mL) was added to a
solution of aldol 10 (400 mg, 1.21 mmol), triphenylphosphine (2.22 g,
8.47 mmol), and p-nitrobenzoic acid (1.42 g, 8.47 mmol) in benzene
(40 mL) at room temperature. The resulting solution was stirred for
17 h at room temperature. The solvent was removed under reduced
pressure, and the reaction crude was purified by flash chromatography
(EtOAc:hexanes 1:7), yielding the nitrobenzoate ester 14 as a colorless
Methyl (2R,3R)-3-Hydroxy-2-methyl-7-octynoate (17). Following
the procedure described above for the preparation of â-hydroxy methyl
ester 16, benzoate ester 15 (273 mg, 0.57 mmol) was treated with 30%
H2O2 (0.25 mL, 2.28 mmol) and LiOH (76 mg, 1.8 mmol). After
workup, the mixture of acids was esterified with excess of diaz-
omethane. Purification gave the â-hydroxy methyl ester 17 as a
colorless viscous oil (77 mg, 74% yield). [R]D25 -2.6 (c 0.5, CHCl3).
(+) FABMS (glycerol matrix) m/z (% relative intensity): 185 [M +
H]+ (74), 167 (5), 153 (20), 125 (7), 107 (44). EIMS m/z (% relative
intensity): 173 (1), 151 (2), 143 (2), 117 (56), 107 (7), 97 (9), 88 (100).
HREIMS calcd for C10H16O3 m/z 184.1099 (M+), found 184.1099. 1H
NMR (300.13 MHz, CDCl3): δ 1.21 (d, J ) 7.2 Hz, 3 H), 1.48-1.80
(m, 4 H), 1.94 (t, J ) 2.6, 1 H), 2.22 (m, 2 H), 2.52 (q, J ) 7.0, 1 H),
2.66 (d, J ) 6.4, 1 H), 3.66 (m, 1 H), 3.70 (s, 3 H). 13C NMR (75.47
MHz, CDCl3): δ 14.7 (c), 18.6 (t), 24.8 (t), 33.9 (t), 45.6 (d), 52.2 (c),
69.0 (d), 73.2 (d), 84.6 (s), 176.8 (s).
Preparation of 3-O-[(-)-(R)-r-Methoxy-r-(9-anthryl)acetyl]-2-
methyloct-7-ynoic Methyl Esters 18-21. General. The esters 18-
21 were prepared by treatment of the corresponding â-hydroxy ester
(3 mg) with (-)-(R)-R-methoxy-R-(9-anthryl)acetic acid (5 mg) in the
presence of DCC (4 mg) and DMAP (catalytic) in CH2Cl2 at room
temperature. After 12 h the solvent was evaporated, and the crude
was sequentially purified by flash chromatography (CH2Cl2) and HPLC
(µ-porasil, 30 cm × 7.8 mm; AcOEt:hexane 7:93; flow rate: 2.0 mL/
min).
25
viscous oil (500 mg, 86%). [R]D +72.1 (c 0.4, CHCl3). EIMS m/z
(% relative intensity): 478 (M+, 2), 382 (1), 327 (1), 311 (5), 302
(11), 296 (10), 283 (2), 272 (4), 244 (27), 231 (3), 178 (5), 150 (100),
135 (51), 134 (22), 117 (22), 104 (28), 91 (38). HREIMS: calcd for
C26H26N2O7 m/z 478.1740 (M+), found 478.1745. FTIR (CHCl3) 3291,
2945, 1780, 1720, 1604, 1528, 1449, 1387, 1350, 1274, 1214, 1110,
1020 cm-1 1H NMR (300.13 MHz, CDCl3): δ 1.33 (d, J ) 7.0 Hz,
.
3 H), 1.66 (m, 2 H), 1.88 (m, 1 H), 1.95 (t, J ) 2.6 Hz, 1 H), 2.05 (m,
1 H), 2.26 (m, 2 H), 2.74 (dd, J ) 13.3, 9.5 Hz, 1 H), 3.27 (dd, J )
13.3, 3.3 Hz, 1 H), 3.97 (t, J ) 8.2 Hz, 1H), 4.12 (dd, J ) 9.1, 2.6 Hz,
1 H), 4.30 (dd, J ) 8.6, 7.0 Hz, 1 H), 4.55 (m, 1 H), 5.55 (ddd, J )
8.2, 8.2, 3.2 Hz, 1 H), 7.14-7.17 (m, 2 H), 7.25-7.33 (m, 3 H), 8.16-
8.20 (m, 2 H), 8.26-8.30 (m, 2 H). 13C NMR (75.47 MHz, CDCl3):
δ 14.6 (c), 18.6 (t), 23.9 (t), 30.7 (t), 38.1 (t), 41.6 (d), 55.6 (d), 66.5
(t), 69.5 (d), 76.3 (d), 83.9 (s), 124.0 (d), 127.9 (d), 129.4 (d), 129.8
(d), 131.2 (d), 135.2 (s), 135.7 (s), 151.0 (s), 153.5 (s), 164.1 (s), 174.2
(s).
(4R,5S,2′R,3′R)-3-[3′-[(4′′-Nitrobenzoyl)oxy]-2′-methyl-7′-octynoyl]-
5-phenyl-4-methyl-2-oxazolidinone (15). Aldol 11 (270 mg, 0.82
mmol) was treated with triphenylphosphine (1.5 g, 5.74 mmol),
p-nitrobenzoic acid (960 mg, 5.74 mmol), and diethylazodicarboxylate
(0.9 mL, 5.74 mmol) in the manner described for the preparation of
nitrobenzoate ester 14. After workup and purification ester 15 was
obtained as an oil together with a small amount of another product
(a) (2S,3S)-3-O-[(-)-(R)-r-methoxy-r-(9-anthryl)acetyl]-2-me-
25
thyloct-7-ynoic Methyl Ester (18). [R]D -119.1 (c 0.2, CHCl3).
EIMS m/z (% relative intensity): 432 (M+, 16), 221 (100), 206 (15),
205 (11), 178 (16). HREIMS calcd for C27H28O5 m/z 432.1936 (M+),
found 432.1963. 1H NMR (300.13 MHz, CDCl3): δ 0.45 (m, 2 H),
1.05 (d, J ) 7.2, 3 H), 1.12 (m, 1 H), 1.29 (m, 1 H), 1.44 (m, 2 H),
1.69 (t, J ) 2.6, 1 H), 2.58, (q, J ) 7.1, 1 H), 3.44 (s, 3 H), 3.59 (s,
3 H), 5.09 (m, 1 H), 6.22 (s, 1 H), 7.44-7.56 (m, 4 H), 8.00-8.03 (m,
2 H), 8.47-8.52 (m, 3 H). 13C NMR (75.47 MHz, CDCl3): δ 12.9
(c), 17.6 (t), 22.8 (t), 29.5 (t), 43.2 (d), 52.1 (c), 57.9 (c), 68.7 (d),
75.1 (d), 77.3 (d), 83.7 (s), 124.7 (d), 125.5 (d), 126.9 (d), 127.6 (s),
129.5 (d), 129.6 (d), 130.9 (s), 131.8 (s), 171.1 (s), 174.0 (s).
(b) (2R,3R)-3-O-[(-)-(R)-r-Methoxy-r-(9-anthryl)acetyl]-2-me-
thyloct-7-ynoic Methyl Ester (19). [R]D25 -87.6 (c 0.2, CHCl3). EIMS
m/z (% relative intensity): 432 (M+, 23), 221 (100), 206 (19), 205
(16), 178 (13). HREIMS calcd for C27H28O5 m/z 432.1936 (M+), found
432.1963. 1H NMR (300.13 MHz, CDCl3): δ 0.42 (d, J ) 7.1, 3 H),
1.52-1.68 (m, 2 H), 1.95 (t, J ) 2.6, 1 H), 2.19, (m, 2 H), 2.33 (q, J
) 7.0, 1 H), 2.94 (s, 3 H), 3.42 (s, 3 H), 5.15 (m, 1 H), 6.26 (s, 1 H),
7.45-7.57 (m, 4 H), 8.00-8.03 (m, 2 H), 8.48-8.57 (m, 3 H). 13C
NMR (75.47 MHz, CDCl3): δ 11.8 (c), 18.4 (t), 24.4 (t), 29.6 (t), 42.7
(d), 51.5 (c), 57.8 (c), 69.2 (d), 75.2 (d), 77.5 (d), 84.0 (s), 124.8 (d),
125.4 (d), 126.9 (d), 127.5 (s), 129.5 (d), 129.6 (d), 130.9 (s), 131.8
(s) 171.0 (s), 173.2 (s).
which could not be separated by HPLC (350 mg, 90% yield). [R]25
D
-8.4 (c 0.7, CHCl3). EIMS m/z (% relative intensity): 478 (M+, 1),
382 (1), 311 (3), 302 (2), 272 (2), 244 (3), 178 (6), 150 (100), 135
(27), 134 (40), 118 (13), 104 (26), 91 (21). HREIMS: calcd for
C26H26N2O7 m/z 478.1740 (M+), found 478.1730. FTIR (CHCl3) 3292,
2942, 2869, 1781, 1720, 1611, 1526, 1453, 1348, 1274, 1233, 1198,
1115, 1078 cm-1 1H NMR (300.13 MHz, CDCl3): δ 0.85 (d, J ) 6.6
.
Hz, 3 H), 0.95 (d, J ) 6.6 Hz, 3 H), 1.61-1.72 (m, 2 H), 1.84-1.95
(m, 1 H), 1.95 (t, J ) 2.7 Hz, 1 H), 2.00-2.08 (m, 1 H), 2.23-2.34
(m, 2 H), 4.29 (d, J ) 6.6 Hz, 1 H), 4.65 (q, J ) 6.8 Hz, 1 H), 5.44
(d, J ) 7.3 Hz, 1 H), 5.55 (td, J ) 8.6, 8.6, 3.3 Hz, 1 H), 7.20-7.31
(m, 2 H), 7.35-7.42 (m, 3 H), 8.27-8.31 (m, 2 H), 8.26-8.30 (m, 2
H). 13C NMR (75.47 MHz, CDCl3): δ 14.5 (c), 18.6 (c), 23.9 (t),
27.4 (t), 30.7 (t), 41.8 (d), 55.3 (d), 69,5 (d), 76.3 (d), 79.3 (d), 83.8
(s), 124.0 (d), 125.9 (d), 129.2 (d), 131.2 (d), 133.3 (d), 135.8 (s), 137.6
(s), 151.0 (s), 153.0 (s), 164.2 (s), 174.2 (s).
Methyl (2S,3S)-3-Hydroxy-2-methyl-7-octynoate (16). A 0 °C
cooled solution of nitrobenzoate ester 14 (414 mg, 0.86 mmol) in THF:

Onchidin B
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11643
(c) (2S,3R)-3-O-[(-)-(R)-r-Methoxy-r-(9-anthryl)acetyl]-2-me-
thyloct-7-ynoic Methyl Ester (20). [R]D25 -74.5 (c 0.1, CHCl3). EIMS
m/z (% relative intensity): 432 (M+, 5), 221 (100), 208 (18), 206 (15),
205 (12), 180 (16), 178 (19), 152 (12). HREIMS calcd for C27H28O5
m/z 432.1936 (M+), found 432.1963. 1H NMR (300.13 MHz,
CDCl3): δ 0.43 (d, J ) 7.1 Hz, 3 H), 1.19-1.67 (m, 4H), 1.96, (t, J
) 2.6 Hz, 1 H), 2.18-2.27 (m, 3 H), 2.94 (s, 3 H), 3.41 (s, 3 H), 5.15
(q, J ) 6 Hz, 1 H), 6.28 (s, 1 H), 7.45-7.57 (m, 4 H), 8.01-8.03 (m,
2 H), 8.48-8.59 (m, 3 H). 13C NMR (75.47 MHz, CDCl3): δ 11.7
(c), 18.5 (t), 24.7 (t), 31.2 (t), 43.0 (d), 51.5 (c), 57.8 (c), 69.3 (d),
75.0 (d), 77.4 (d), 84.1 (s), 124.9 (d), 125.4 (d), 126.8 (d), 127.3 (s),
129.5 (d), 129.6 (d), 130.9 (s), 131.8 (s) 170.9 (s), 173.5 (s).
Determination of Absolute Configuration of Hymo (5). Onchidin
B (0.5 mg) was submitted to hydrolysis with 6 M HCl (0.5 mL) in a
sealed tube at 110 °C for 24 h. The excess HCl was removed by passing
a stream of N2, and the residue was dried under vacuum. The
hydrolyzates were diluted with water (1 mL), and the hydroxy acids
were extracted with diethyl ether (3 × 1 mL). The ethereal solution
was dried under vacuum, and the residue was dissolved in 1 mL of
ether and treated with diazomethane for 30 min. The excess of reagent
was removed with a stream of dry N2. The hydroxy methyl ester was
treated with (-)-(R)-R-methoxy-R-(9-anthryl)acetic acid (0.5 mg), DCC
(0.5 mg), and DMAP (catalytic) in CH2Cl2 at room temperature. After
12 h the solvent was evaporated, and the residue was subjected to
HPLC-MS (µ-porasil, 30 cm × 7.8 mm; AcOEt:hexane 7:93; flow
rate: 2.0 mL/min) to show peak at tR 30.69 min.
(d) (2R,3S)-3-O-[(-)-(R)-r-Methoxy-r-(9-anthryl)acetyl]-2-me-
thyloct-7-ynoic Methyl Ester (21). [R]D25 +20.1 (c 0.1, CHCl3). EIMS
m/z (% relative intensity): 432 (M+, 18), 221 (100), 206 (14), 205 (9),
Acknowledgment. This work was financially supported by
grants from CICYT (MAR95-1933-CO2-O2 and PM95-0135)
and the Xunta de Galicia (XUGA-20907B94 and 20901B95).
R.F. acknowledges a fellowship from the Xunta de Galicia. We
are grateful to the Mass Spectrometry Laboratory, University
of Illinois, School of Chemical Sciences, for the tandem
FABMS/MS.
1
178 (18). HREIMS m/z 432.1963 for C27H28O5 (calcd 432.1936). H
NMR (300.13 MHz, CDCl3): δ 0.50 (m, 2 H), 1.01 (d, J ) 7.0, 3 H),
1.08 (m, 1 H), 1.22-1.30 (m, 1 H), 1.39-1.53 (m, 2 H), 1.69 (t, J )
2.6, 1 H), 2.55, (dd, J ) 7.1, 5.2, 1 H), 3.44 (s, 3 H), 3.64 (s, 3 H),
5.13 (m, 1 H), 6.26 (s, 1 H), 7.45-7.56 (m, 4 H), 8.00-8.03 (m, 2 H),
8.47-8.54 (m, 3 H). 13C NMR (75.47 MHz, CDCl3): δ 12.4 (c), 17.9
(t), 23.7 (t), 30.7 (t), 43.5 (d), 52.6 (c), 58.2 (c), 69.0 (d), 74.9 (d),
77.8 (d), 84.0 (s), 124.7 (d), 125.7 (d), 127.2 (d), 127.8 (s), 129.8 (d),
129.9 (d), 131.0 (s), 132.0 (s), 171.5 (s), 174.6 (s).
(e) Separation of the Mixture of 3-O-[(-)-(R)-r-Methoxy-r-(9-
anthryl)acetyl]-2-methyloct-7-ynoic Methyl Esters (18-21) by HPLC-
MS. A mixture of the synthetic isomers was separated by HPLC-MS
(µ-porasil, 30 cm × 7.8 mm; AcOEt:hexane 7:93; flow rate: 2.0 mL/
min) showing the following retention times: (S,R) 28.09 min, (R,R)
30.93 min, (R,S) 41.74 min, and (S,S) 46.54 min.
Supporting Information Available: 1H NMR, 13C NMR,
COSY, TOCSY, HMQC, ROESY spectra, and expansions of
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