Isolation and structure determination of obyanamide, a novel cytotoxic cyclic depsipeptide from the marine cyanobacterium Lyngbya confervoides

Article abstract of DOI:10.1021/np0102253

Obyanamide (1) was isolated from a variety of the marine cyanobacterium Lyngbya confervoides collected in Saipan, Commonwealth of the Northern Mariana Islands. Gross structure elucidation of this novel cyclic depsipeptide relied on extensive application of 2D NMR techniques. The absolute stereochemistry was deduced by chiral chromatography of the hydrolysis products and comparison with authentic and synthetic standards. Obyanamide (1) was cytotoxic against KB cells with an IC₅₀ of 0.58 μg/mL.

Full text of DOI:10.1021/np0102253

J . Nat. Prod. 2002, 65, 29-31
29
Isola tion a n d Str u ctu r e Deter m in a tion of Obya n a m id e, a Novel Cytotoxic Cyclic
Dep sip ep tid e fr om th e Ma r in e Cya n oba cter iu m Lyn gbya con fer void es
Philip G. Williams,† Wesley Y. Yoshida,† Richard E. Moore,*,† and Valerie J . Paul*,‡
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, and
University of Guam Marine Laboratory, UOG Station, Mangilao, Guam 96913
Received May 3, 2001
Obyanamide (1) was isolated from a variety of the marine cyanobacterium Lyngbya confervoides collected
in Saipan, Commonwealth of the Northern Mariana Islands. Gross structure elucidation of this novel
cyclic depsipeptide relied on extensive application of 2D NMR techniques. The absolute stereochemistry
was deduced by chiral chromatography of the hydrolysis products and comparison with authentic and
synthetic standards. Obyanamide (1) was cytotoxic against KB cells with an IC50 of 0.58 µg/mL.
1
Cyanobacteria are an ancient and diverse group of
microorganisms. They are able to inhabit and thrive in an
incredible variety of environments, in part because of their
ability to produce a rich range of secondary metabolites.
These metabolites have already been shown to display a
wide spectrum of biological activities ranging from anti-
bacterial1 to immunosuppressive.2 This diversity has gen-
erated considerable interest in cyanobacteria, since over
half of the new drugs approved from 1983 to 1994 and 60%
of the drugs currently approved for the treatment of cancer
are still of natural origin.3 One compound already in clinical
trials for the treatment of cancer is based on the crypto-
phycins4 isolated from terrestrial cyanobacteria. Some of
the most common and accessible marine cyanobacteria in
the subtropical and tropical oceans belong to the species
Lyngbya.5 Our ongoing investigation of cyanobacteria has
led to the isolation of obyanamide (1) from a collection of
Lyngbya confervoides.
degrees of unsaturation. From H and 13C NMR spectrum
analysis, it was evident that 1 contained 14 sp2 carbons in
the form of four carbon carbon double bonds and six carbon
heteroatom double bonds, which accounted for 10 degrees
of the unsaturation. The remaining three degrees of
unsaturation must then be in the form of rings. The IR
spectrum revealed that 1 contained both amide (1628 cm-1
)
and ester bonds (1734 cm-1), indicating it was in fact a
peptolide. This peptidic nature was further supported by
the presence of two amide (δ 9.11, 7.90) and two N-
methylamide proton signals (δ 3.085, 3.094) in the 1H NMR
spectrum. The COSY, HSQC, and HMBC data, sum-
marized in Table 1, suggested the presence of N-methylva-
line, N-methylphenylalanine, and 3-aminopentanoic acid
(Apa) fragments. The low-field resonance for C-29 (δ 67.9)
and the corresponding proton (δ 5.20) indicated that the
acyloxy group was attached to this carbon. A COSY
correlation from H-30 and a HMBC cross-peak from C-1
to H-29 along with a HMBC correlation from C-28 to H-30
expanded this particular fragment into a lactic acid moiety,
which was attached to the â-amino acid (Apa) unit via an
ester linkage. The downfield singlet in the 1H NMR
spectrum at δ 7.98 as well as the sp2 carbon resonances at
123.2, 149.0, 160.4, and 170.1 ppm were characteristic of
a 2-alkylthiazole-4-carboxylic acid unit (C-6 to C-9).8 HMBC
correlations from C-9 and C-10 to H-11 and from C-9 to
H-8 connected the remaining carbons to form an Ala-
thiazole fragment.
Resu lts a n d Discu ssion
The lipophilic extract of VP680 was cytotoxic and showed
slight solid tumor selectivity in the Corbett assay.6,7 Solvent
partitioning of this extract followed by Si gel chromatog-
raphy and repeated reversed-phase HPLC yielded 1 as a
white amorphous powder. Obyanamide (1) exhibited mod-
erate cytotoxicity against KB and LoVo cells with IC50
values of 0.58 and 3.14 µg/mL, respectively.
As always, HMBC couplings to the N-methyl groups
were quite informative in sequencing the peptolide. To the
N-methyl signal at 3.085 ppm (H-21), HMBC cross-peaks
from the signals for the R carbon of phenylalanine (C-13)
and the carbonyl carbon of valine (C-22) were visible. This
clearly indicated that the N-Me-Phe nitrogen was attached
to the N-Me-Val carbonyl. Similarly the N-methyl signal
at δ 3.094 (H-27) showed HMBC correlations from the R
carbon of the valine (C-23) and the carbonyl carbon of the
lactic acid fragment (C-28). Finally, a HMBC correlation
between the δ 7.90 amide proton of the Ala-thiazole moiety
and C-12 of the N-Me-Phe residue firmly established the
connection of the Ala nitrogen to the N-Me-Phe carbonyl.
Since one degree of unsaturation remained, 1 had to be
cyclic with the amine of the Apa attached to the cysteine-
derived carbonyl of the thiazole ring (C-6).
Gr oss Str u ctu r e. HRFABMS analysis established the
molecular formula for 1 as C30H41N5O6S, indicating 13
Absolu te Ster eoch em istr y. The absolute stereochem-
istry of 1 was established by analysis of the degradation
products. Obyanamide was subjected to ozonolysis followed
by acid hydrolysis, and the product mixture was analyzed
* To whom correspondence should be addressed. (R.E.M.) Tel: (808) 956-
7232. Fax: (808) 956-5908. E-mail: moore@gold.chem.hawaii.edu. (V.J .P.)
Tel: (671) 735-2186. Fax: (671) 734-6767. E-mail: vpaul@uog9.uog.edu.
† University of Hawaii at Manoa.
‡ University of Guam Marine Laboratory.
10.1021/np0102253 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/21/2001

30 J ournal of Natural Products, 2002, Vol. 65, No. 1
Williams et al.
Ta ble 1. NMR Spectral Data for Obyanamide (1) in CDCl3
1H-1H COSYa
HMBCa,c
b
C/H no.
δHa (J in Hz)
δC
3-Aminopentanoic Acid
170.4
2.74, dd (-11.9, 39.1 H-3
1
2
H-2, H-29
5.0)
2.38, dd (-11.9,
3.0)
3
4.36, m
9.11, d (10.4)
1.63, m
47.6 H-2, 3-NH, H-4
H-3
26.2 H-3, H-5
11.3 H-4
H-4, H-5
3-NH
4
5
H-2, H-5
H-4
1.05, t (7.4)
F igu r e 1. Synthesis of (R)-3-aminopentanoic acid.
Alanine-Thiazole
160.4e
149.0
123.2
170.1
acid. â-Amino acid fragments are not uncommon in marine
metabolites found in cyanobacteria and certain sea hares.
Structurally related units include the (2S,3R)-3-amino-2-
methylpentanoic acid found in majusculamide C,10 dolas-
tatin 11,11 dolastatin 12,11 and lyngbyastatin 1.12 Similarly,
dolastatin 1613 possesses a 3-amino-2,4-dimethylpentanoic
acid fragment of unknown stereochemistry, while dolas-
tatin D14 has a (2R,3R)-3-amino-2-methylbutanoic acid.
6
7
8
9
H-8e
H-8
7.98, s
H-8, H-11,
10-NHe
H-11, 10-NH
10
5.04, dq (6.8,
5.9)
48.3 H-11
10-NH 7.90, d (5.9)
11
1.42, d (6.8)
24.1 H-10
H-10
N-Me Phenylalanine
168.2
12
13
H-14, 10-NH
H-14, H-21
Exp er im en ta l Section
5.42, dd (8.4,
6.7)
60.9 H-14
Gen er a l Exp er im en ta l P r oced u r es. The UV spectra was
determined on a Hewlett-Packard 8453 spectrophotometer,
and the IR spectra were recorded on a Perkin-Elmer 1600
FTIR instrument as a film on a NaCl disk. The optical rotation
was measured on a J asco-DIP-700 polarimeter at the sodium
D line (589 nm). FABMS and HRFABMS were recorded in the
positive mode on a VG ZAB2SE spectrometer. The NMR
spectra of 1 were recorded in CDCl3 on a Varian Unity Inova
400wb at 400 MHz and a GE GN Omega 500 operating at 500
MHz using the residual solvent signal as internal reference.
NMR analyses of the synthetic products were carried out at
300 and 75 MHz using a GE QE-300 spectrometer. HPLC
separations were performed on a Beckman 110B apparatus
coupled to a Applied Biosystems 759A absorbance detector.
All synthetic reagents and amino acids were purchased from
Aldrich.
Biologica l Ma ter ia l. The dark reddish-black clumps of
cyanobacterium were collected at Obyan Bay in Saipan and
designated VP680. A voucher is maintained in formalin at the
University of Guam Marine Laboratory. The sample was
identified by V. J . Paul and E. Cruz-Rivera.
Extr a ction a n d Isola tion of Obya n a m id e (1). VP680
was extracted with 1:1 CH2Cl2-MeOH to yield 1.50 g of
lipophilic extract. This extract was then partitioned between
hexane and 80% MeOH. After drying, the aqueous methanol
residue was partitioned between water and n-butanol to afford
154 mg of material from the organic layer. Separation on a
14
3.25, dd (-13.7, 37.2 H-13
8.4)
2.87, dd (-13.7,
6.7)
15
16, 20
17, 19
137.0
128.9 H-17
129.4 H-16, H-18
H-14
H-14
7.17, d (7.9)
7.21, dd (7.9,
7.2)
7.08, t (7.2)
3.085, s
18
21
127.1 H-17
29.1
H-16
H-13
N-Me Valine
169.8
22
23
H-21,H-23,
H-13
H-26, H-25,
H-27
5.06, d (10.2)
57.9 H-24
24
25
26
27
2.28, m
27.6 H-23, H-25, H-26 H-23
0.84, d (6.6)
0.48, d (6.6)
3.094, s
18.5 H-24
18.6 H-24
29.9
H-26
H-25
H-23
Lactic Acid
173.2
67.9 H-30
16.0 H-29
28
29
30
H-27, H-30
H-30
H-29
5.20, q (6.9)
1.23, d (6.9)
a
b
Recorded at 400 MHz. Recorded at 100 MHz. c Protons
d
showing long-range correlation with indicated carbon. If not
indicated otherwise, correlations were observed for J CH ) 7 Hz.
n
e Recorded at 125 MHz.
C
18 column with increasing amounts of MeCN in H2O resulted
by HPLC on a chiral column [Chirex phase 3126 (D)]. A
comparison with authentic amino acid standards estab-
lished the configurations of N-Me-Phe, N-Me-Val, and Ala
as L. Pure L and D lactic acid standards were prepared from
alanine,9 and the S stereochemistry at C-29 was deter-
mined by chiral TLC and chiral HPLC. Due to the limited
amount of material available, determination of the final
stereocenter at C-3 necessitated the synthesis of (R)- and
(S)-3-aminopentanoic acid (Figure 1). Commercially avail-
able (R)- and (S)-2-aminobutanol served as the chiral
starting material for a five-step synthesis. Boc protection
and tosylation of the amino alcohol followed by sodium
cyanide substitution and acid hydrolysis provided the
necessary standards. A comparison of the hydrolyzate
mixture and these standards showed no sign of the R-Apa
under a variety of solvent strengths and flow rates.
Conversely a peak coeluting with the S-Apa standard was
clearly evident.
in the activity concentrated primarily in the 60% MeCN in
H2O fraction (9 mg). Further purification on a RP column (50%,
60%, 70% MeCN in H2O) gave 4.7 mg of material in the 60%
MeCN fraction that after semipreparative RP HPLC (Ultra-
carb, 5 ODS 30, 10 × 250 mm, 65% CH3CN in H2O, 3 mL/
min, detection at 220 nm) led to 1.6 mg of crude 1 (tR ) 16
min). Final HPLC purification with 70% MeOH (tR ) 13.8 min)
yielded 1.1 mg of pure 1.
Obyanamide (1) was obtained as a white powder: [R]27
D
+20° (c 0.04, MeOH); UV (MeOH) λmax (log ꢀ) 210 (5.20), 222
(4.32) nm; IR (film) νmax 3315, 1734, 1628, 1551, 1521, 1458,
1279, 699 cm-1; 1H NMR, 13C NMR, 1H-1H COSY, and HMBC
data, see Table 1; FABMS m/z 622.4 [M + Na]+; HRFABMS
m/z [M + H]+ 600.2873 (calcd for C30H42N5O6S, 600.2859).
Syn th esis of 3-Am in op en ta n oic Acid Sta n d a r d s: (R)-
N-Boc-2-a m in obu ta n ol (2).15 To a 5 mL vial containing 100
µL of (R)-2-aminobutanol was added 50 mg of NaOH (1 molar
equiv) in 1 mL of water and 750 µL of tert-butyl alcohol. Next
230 mg of di-tert-butyl carbonate was added and the cloudy
solution stirred overnight at room temperature. The next day
the solution was adjusted to pH 7 with 6 N HCl and partitioned
between ethyl acetate and water. The organic phase was dried
Obyanamide (1) is a novel depsipeptide containing 2
N-methyl amino acids, an Ala-thiazole unit, and a â-amino

Cytotoxic Cyclic Depsipeptide from Lyngbya
J ournal of Natural Products, 2002, Vol. 65, No. 1 31
over MgSO4 and the solvent removed in vacuo to yield 151
mg (69%) of compound 2: 1H NMR (CDCl3) δ (carbon position,
integration, multiplicity; J in Hz) 0.80 (4, 3H, t; 7.87), 1.30
(7/8/9, 9H, s), 1.42 (3, 2H, m), 3.38 (2, 1H, m), 3.42 (1, 2H, m),
5.07 (OH, 1H, br. s); 13C NMR δ 64.0 (1), 53.8 (2), 24.3 (3),
10.2 (4), 156.7 (5), 79.6 (6), 28.2 (7/8/9).
HPLC, comparing the retention times of the components of
the hydrolyzate with those of authentic standards [Column
Chirex Phase 3126 (D) (4.6 × 250 mm), Phenomenex; solvent
2 mM CuSO4 for Ala and N-Me-Val, CuSO4-MeCN (95:5) for
lactic acid and 3-aminopentanoic acid and CuSO4-MeCN (85:
15) for N-Me-Phe, flow rate 1 mL/min, except alanine 0.8 mL/
min; detection at 254 nm]. The retention times (tR, min) of the
standards were L-Ala (12.1), D-Ala (16.8), N-Me-L-Val (20.5),
N-Me-D-Val (26.5), L-lactic acid (18.5), D-lactic acid (31.5),
N-Me-L-Phe (28.3), N-Me-D-Phe (29.9), (S)-3-aminopentanoic
acid (8.2), and (R)-3-aminopentanoic acid (13.0). The retention
times of the amino acid components of the hydrolyzate were
(S)-3-aminopentanoic acid (8.0), L-Ala (11.8), L-lactic acid
(18.5), N-Me-L-Val (20.5), and N-Me-L-Phe (28.3). The identi-
ties of the peaks were also confirmed by co-injection.
(R)-N-Boc-2-a m in obu ta n ol Tosyla te (3).16 A 151 mg
sample of 2 was dissolved in 1 mL of freshly distilled pyridine,
and the solution was stirred at 0 °C while 293 mg of
toluenesulfonyl chloride (2 molar equiv) was slowly added. The
reaction mixture was stored at -4 °C for 3 days and then
poured over 30 mL of ice in a 60 mL separatory funnel and
extracted with 30 mL of diethyl ether. The organic layer was
subsequently washed with 6 N HCl followed by saturated
aqueous NaHCO3 and saturated aqueous sodium chloride. The
organic layer was dried over MgSO4 and evaporated to yield
263 mg (100%) of 3: 1H NMR (CDCl3) δ (carbon position,
integration, multiplicity; J in Hz) 0.79 (4, 3H, t; 7.3), 1.32 (7/
8/9, 9H, s), 1.40 (3, 2H, m), 2.36 (16, 3H, s), 3.49 (2, 1H, m)
3.94 (1, 2H, m), 7.70 (12/14, 2H, d, 7.1), 7.23 (11/15, 2H, d;
7.8); 13C NMR δ (carbon position) 71.3 (1), 65.9 (2), 24.4 (3),
10.4 (4), 155.3 (5), 79.6 (6), 28.5 (7/8/9), 145.0 (10), 128.0 (11/
15), 130.0 (12/14), 158.3 (13), 21.8 (16).
(R)-N-Boc-3-a m in op en ta n itr ile (4). To 250 mg of 3 in 2
mL of wet DMSO was added 43 mg of NaCN with stirring.
The reaction was left at room temperature overnight and then
partitioned between diethyl ether and water. The ether layer
was evaporated and the residue chromatographed on silica gel
using 10:1 hexane-ethyl acetate to afford 66 mg (44%) of 4:
1H NMR (CDCl3) δ (carbon position, integration, multiplicity;
J in Hz) 0.90 (5, 3H, t; 7.3), 1.39 (8/9/10, 9H, s), 1.49 (4, 2H,
m), 2.46 (2, 1H, dd; -16.1, 5.1), 2.68 (2, 1H, dd; -16.1, 4.6),
3.62 (3, 1H, m); 13C NMR δ (carbon position) 117.4 (1), 40.9
(2), 49.0 (3), 23.5 (4), 10.4 (5), 155.3 (6), 79.9 (7), 28.4 (8/9/10).
(R)-3-Am in op en ta n itr ile (5).9 A solution of 66 mg (0.3
mmol) of 4 in 1 mL of concentrated trifluoroacetic acid was
stirred for 10 min. Evaporation of the solvent under N2 and
partitioning of the residue between EtOAc and water yielded
30 mg (92%) of 5 from the aqueous phase: 1H NMR (MeOH-
d4) δ (carbon position, integration, multiplicity; J in Hz) 1.09
(5, 3H, t; 7.6), 1.85 (4, 2H, m), 2.98 (2, 2H, d; 5.6), 3.51 (3, 1H,
m); 13C NMR δ (carbon position) 115.8 (1), 25.4 (2), 49.3 (3),
20.3 (4), 8.5 (5).
Ack n ow led gm en t. This research was supported by NC-
NPDDG grant CA53001 from the National Cancer Institute.
We are grateful to the Commonwealth of the Northern
Marianas for a marine research permit. J ohn Starmer collected
the cyanobacterium, and Edwin Cruz-Rivera assisted in the
identification. Dr. M. Lieberman and G. Tien, University of
Hawaii, Department of Chemistry, carried out bioassays. The
UCR Mass Spectrometry Facility, Department of Chemistry,
University of California, performed mass spectral analyses at
Riverside.
Refer en ces a n d Notes
(1) Sticher, O.; Heilmann, J .; J aki, B. J . Nat. Prod. 2000, 63, 1283-1285.
(2) Koehn, F. E.; Longley, R. E.; Reed, J . K. J . Nat. Prod. 1992, 55, 613-
619.
(3) Cragg, G. M.; Newman, D. J .; Snader, K. M. J . Nat. Prod. 1997, 60,
52-60.
(4) Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.; Corbett,
T. H.; Valeriote, F. A.; Demchik, L. J . Am. Chem. Soc. 1994, 116,
4729-4737.
(5) Moore, R. E. Pure Appl. Chem. 1982, 54, 1919-1934.
(6) Corbett, T. H.; Valeriote, F. A.; Polin, L.; Panchapor, C.; Pugh, S.;
White, K.; Lowichik, N.; Knight, J .; Bissery, M.-C.; Wozniak, A.;
LoRusso, P.; Biernat, L.; Polin, D.; Knight, L.; Biggar, S.; Looney,
D.; Demchik, L.; J ones J .; J ones, L.; Blair, S.; Palmer, K.; Essenma-
cher, S.; Lisow, L.; Mattes, K. C.; Cavanaugh, P. F.; Rake, J . B.; Baker,
L. In Cytotoxic Anticancer Drugs: Models and Concepts for Drug
Discovery and Development; Valeriote, F. A., Corbett, T. H., Baker,
L. H., Eds.; Kluwer Academic Publishers: Norwell, 1992; pp 35-87.
(7) After NMR characterization, FABMS indicated a second compound
was present in the sample (FABMS m/z ) 1408). Further HPLC
purification using MeOH yielded pure 1 as described above, but it
was this other minor compound which was responsible for most of
the activity. Unfortunately too little material was isolated (<200 µg)
for characterization of the compound, which exhibited cytotoxicity
<1 µg/mL against the KB cell line.
(8) For representative examples see: (a) Sone, H.; Kigoshi, H.; Yamada,
K. Tetrahedron 1997, 53, 8149-8154. (b) Luesch, H.; Yoshida, W.
Y.; Moore, R. E.; Paul, V. J .; Mooberry, S. L. J . Nat. Prod. 2000, 63,
611-615. (c) Pettit, G. R.; Kamano, Y.; Brown, P.; Gust, D.; Inoue,
M.; Herald, C. L. J . Am. Chem. Soc. 1982, 104, 905-907. (d) Ojika,
M.; Nemoto, T.; Nakmura, M.; Yamada, K. Tetrahedron Lett. 1995,
36, 5057-5058.
(R)-3-Am in op en ta n oic Acid Hyd r och lor id e (6). A solu-
tion of 30 mg of 5 in 1 mL of 6 N HCl was refluxed overnight.
Purification over DOWEX 50 resin eluting with 1 M HCl
resulted in 40 mg (87%) of 6: IR (Nujol) νmax 3415 (s), 3300
(s), 3100 (br), 1712 (s); H NMR (MeOH) δ (carbon position,
integration, multiplicity; J in Hz) 0.98 (5, 3H, t; 7.8), 1.72 (4,
2H, m), 2.69 (2, 1H, dd; -17.3, 4.7), 2.84 (2, 1H, dd; -17.3,
8.3), 3.6 (3, 1H, m), 7.01,7.19, 7.36 (NH3).
(S)-3-Am in open ta n oic Acid Hyd r och lor id e. The S enan-
tiomer was synthesized in the same manner as described above
starting with (S)-2-aminobutanol.
L a n d D-La ctic Acid .9 A solution of L-Ala (100 mg) in 1
mL of 4 N HCl at 4 °C was treated with excess sodium nitrite
(500 mg in 1 mL of water) and left to stir overnight. The
mixture was repeatedly extracted with ethyl ether and evapo-
rated to dryness to give L-lactic acid. D-Lactic acid was
prepared in the same manner from the enantiomer.
(9) Golakoti, T.; Ogino, J .; Heltzel, C. E.; Husebo, T. L.; J ensen, C. M.;
Larsen, L. K.; Patterson, G. M. L.; Moore, R. E.; Mooberry, S. L.;
Corbett, T. H.; Valeriote, F. A. J . Am. Chem. Soc. 1995, 117, 12030-
12049.
(10) Carter, D. C.; Moore, R. E.; Mynderse, J . S.; Niemczura, W. P.; Todd,
J . S. J . Org. Chem. 1984, 49, 236-241.
Ch ir a l TLC a n a lysis. The acid hydrolyzate of 1 was
subjected to TLC analysis on Chiralplate (Macherey-Nagel)
using 1:9 MeOH-CH2Cl2 as the developing solvent. With V2O5
spray reagent17 the lactic acid was visualized as an intense
blue spot. Authentic L-lactic acid and D-lactic acid showed Rf
values of 0.63 and 0.60, respectively. The L-lactic acid in the
hydrolyzate had an Rf of 0.63.
Ozon olysis a n d Acid Hyd r olysis of Obya n a m id e (1).
For analysis, 200 µg of 1 was dissolved in 1 mL of CH2Cl2 and
ozonized for 15 min. The residue was dissolve in 400 µL of 6
N HCl and refluxed at 118 °C for 16 h. After evaporation of
the solvent the sample was passed over a C18 column (100 mg)
with 10% CH3CN.
(11) Pettit, G. R.; Kamano, Y.; Kizu, H.; Dufresne, C.; Herald, C. L.;
Bontems, R. J .; Schmidt, J . M.; Boettner, F. E.; Nieman, R. A.
Heterocycles 1989, 28, 553-558.
(12) Harrigan, G. G.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.; Park, P.
U.; Biggs, J .; Paul, V. J .; Mooberry, S. L.; Corbett, T. H.; Valeriote,
F. A. J . Nat. Prod. 1998, 61, 1221-1225.
(13) Pettit, G. R.; Xu, J .; Hogan, F.; Williams, M. D.; Doubek, D. L.;
Schmidt, J . M.; Cerny, R. L.; Boyd, M. R. J . Nat. Prod. 1997, 60, 752-
754.
(14) Sone, H.; Nemoto, T.; Ishiwata, H.; Ojika, M.; Yamada, K. Tetrahedron
Lett. 1993, 34, 8449-8452.
(15) Kocienski, P. S. Protecting Groups; Gutmann &Co GmbH: New York,
1994; pp 193-5.
(16) Tietze, L. Reactions and Synthesis in the Organic Chemistry Labora-
tory; University Science Books; Mill Valley, CA, 1989; pp 437-438.
(17) Klaus, R.; Fisher, W. Chromatographia 1989, 23, 137-140.
Absolu te Ster eoch em istr y of th e Am in o Acid Der ived
Un its. After hydrolysis the mixture was analyzed by Chiral
NP0102253