Isolation and Structure Determination of Lyngbyastatin 3, a Lyngbyastatin 1 Homologue from the Marine Cyanobacterium Lyngbya majuscula. Determination of the Configuration of the 4-Amino-2,2-dimethyl-3-oxopentanoic Acid Unit in Majusculamide C, Dolastatin 12, Lyngbyastatin 1, and Lyngbyastatin 3 from Cyanobacteria

Article abstract of DOI:10.1021/np0302145

The structure of lyngbyastatin 3 (1), including the configurations of the two unusual amino acid residues, viz., the 3-amino-2-methylhexanoic acid (Amha) and 4-amino-2,2-dimethyl-3-oxopentanoic acid units (Ibu), has been established by chemical degradation. Analysis of the cyanobacterial samples of lyngbyastatin 3 (1), lyngbyastatin 1 (2), and dolastatin 12 (3) demonstrated that they are mixtures of Ibu epimers [R (major) and S (minor)], whereas the structurally related majusculamide C (4) is a single diastereomer having an S-Ibu unit.

Full text of DOI:10.1021/np0302145

1356
J . Nat. Prod. 2003, 66, 1356-1363
Isola tion a n d Str u ctu r e Deter m in a tion of Lyn gbya sta tin 3, a Lyn gbya sta tin 1
Hom ologu e fr om th e Ma r in e Cya n oba cter iu m Lyn gbya m a ju scu la .
Deter m in a tion of th e Con figu r a tion of th e 4-Am in o-2,2-d im eth yl-3-oxop en ta n oic
Acid Un it in Ma ju scu la m id e C, Dola sta tin 12, Lyn gbya sta tin 1, a n d
Lyn gbya sta tin 3 fr om Cya n oba cter ia
Philip G. Williams,† 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 14, 2003
The structure of lyngbyastatin 3 (1), including the configurations of the two unusual amino acid residues,
viz., the 3-amino-2-methylhexanoic acid (Amha) and 4-amino-2,2-dimethyl-3-oxopentanoic acid units (Ibu),
has been established by chemical degradation. Analysis of the cyanobacterial samples of lyngbyastatin
3 (1), lyngbyastatin 1 (2), and dolastatin 12 (3) demonstrated that they are mixtures of Ibu epimers [R
(major) and S (minor)], whereas the structurally related majusculamide C (4) is a single diastereomer
having an S-Ibu unit.
Isolation of the same secondary metabolite from a variety
of organisms that have a dietary or a symbiotic relationship
can cloud the issue of the compound’s true origin. For
example, the dolastatins are a remarkable series of cyto-
toxic peptides and depsipeptides that were originally
isolated from the mollusk Dolabella auricularia.1 The most
potent cytotoxin of the series, dolastatin 10, has been
recently isolated from the cyanobacterium Symploca sp. in
a yield several orders of magnitude greater than what the
extract of the sea hare provided.2,3 This dramatic difference
suggested that the mollusk, a generalist herbivore, may
be acquiring these cytotoxins from cyanobacteria through
diet. The isolation of dolastatins 3, 12 (3), and 16 and a
variety of dolastatin analogues from cyanobacteria lends
further credibility to this proposal.4-7
lyngbyastatin 1 (2) homologue from Lyngbya majuscula
Harvey ex Gomont11 (Oscillatoriaceae) strains collected in
Guam. We report here the isolation and characterization
of 1 along with the configuration of the 4-amino-2,2-
dimethyl-3-oxopentanoic acid (Ibu) unit in 1. In light of the
result, we have also reexamined the configuration of the
Ibu unit in our cyanobacterial samples of lyngbyastatin 1
(2), dolastatin 12 (3), and majusculamide C (4).
In 1998, we reported the isolation and biological evalu-
ation of samples of lyngbyastatin 1 (2) and dolastatin 12
(3) from a Lyngbya majuscula/ Schizothrix calcicola as-
semblage.5 The structure determinations of these cyano-
bacterial samples were hampered by the extensive signal
doubling and broadening observed in the NMR spectra, a
phenomenon not seen in the NMR spectra of majuscula-
mide C (4)7d from cyanobacteria or the samples of dolas-
tatin 11 (5)8 and dolastatin 12 (3)8 from the sea hare. On
the basis of epimerization experiments on 4, we concluded
that the unusual spectral broadness observed in the
cyanobacterial samples of 2 and 3 was due to these samples
being mixtures of diastereomers arising from epimerization
of the acid-sensitive Ibu unit. Synthetic studies have now
established an S configuration for the Ibu unit of dolastatin
119 (5) and more recently have suggested that the Ibu units
of the cyanobacterial metabolites 2 and 3 have an R
configuration. In other words, R-Ibu-3 had been isolated
from the cyanobacteria, whereas S-Ibu-3 had been isolated
from a sea hare.10
Resu lts a n d Discu ssion
Lyngbyastatin 3 (1) was isolated by bioassay-guided
fractionation of the weakly solid tumor selective12
and
highly cytotoxic (even at 1/100 dilution) extracts of NIH143,
-154, and -199. The pure compound 1 had IC50 values of
32 and 400 nM against KB and LoVo cell lines, respec-
tively, but when tested in vivo against colon adenocarci-
noma #38 or mammary adenocarcinoma #16/C in mice, 1
was poorly tolerated and exhibited only marginal or nil
antitumor activity.
We have investigated this problem further as a result
of the structure elucidation of lyngbyastatin 3 (1), a new
* To whom correspondence should be addressed. Tel: (808) 956-7232.
Fax: (808) 956-5908. E-mail: moore@gold.chem.hawaii.edu.
† University of Hawaii at Manoa.
Very few structural features could be elucidated from
the spectral data of 1.
‡ University of Guam Marine Laboratory.
§ Present address: Smithsonian Marine Station, Fort Pierce, FL 34949.
10.1021/np0302145 CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/07/2003

Lyngbyastatin 3 from Lyngbya majuscula
J ournal of Natural Products, 2003, Vol. 66, No. 10 1357
Sch em e 1. Synthesis of Amha Standardsa
a
(i) n-BuLi, THF, (R)-N-benzyl-N-methylbenzylamine; (ii) KHMDS, Mel;
(iii) H2, 20% Pd/C; (iv) 6 N HCl.
the side chain was still intact. Analysis of the hydrolysate
revealed a derivative (7, m/z 333) in which all fragments
derived from cleavage adjacent to the amide linkage were
accounted for (9, 12, and 14). Two new ions, at m/z 274
(10) and 218 (13), established that the extra methylene was
part of the aliphatic side chain of a 3-amino-2-methylhex-
anoic acid (Amha) unit, which has also been found in three
other marine natural products, viz., malevamide B,20
ulongamides A-F,21 and kulokekahilide-1.22
The stereochemistry of 1 was determined by a combina-
tion of chiral HPLC and Marfey analysis.23 Comparison
with authentic standards by chiral HPLC established the
configuration of the R-amino and R-hydroxy acids in 1 as
N,O-dimethyl-L-tyrosine, N-methyl-L-valine, N-methyl-L-
leucine, N-methyl-L-alanine, and L-2-hydroxy-3-methylva-
leric acid. Synthetic standards of the Amha unit were
prepared following Davies’ procedure for anti-R-alkyl-â-
amino acids.24 The Michael addition of (R)-(+)-N-benzyl-
N-R-methylbenzylamine to ethyl hex-2(E)-enoate afforded
the enantiomerically pure adduct 15 in 50% yield (Scheme
1). Treatment of 15 with potassium hexamethyldisilazane
(KHMDS) and an excess of iodomethane gave 16, which
was then hydrogenated and hydrolyzed to afford a mixture
of (2S,3R)- and (2R,3R)-17. Half of this mixture was then
separated as their L-(1-fluoro-2,4-dinitrophenyl)-5-leucina-
mide (L-FDLA)25 derivatives by HPLC. The configurations
of these standards were determined on the basis of the ratio
of the derivatives with the major peak being assigned as
L-FDLA-(2R,3R)-17 as expected from synthesis.24 Deriva-
tization of the remaining amount of 17 with DL-FDLA and
subsequent HPLC separation produced standards equiva-
lent to (2R,3S)- and (2S,3S)-17.23,26,27 Comparison by LC-
MS of these standards with the L-FDLA-derivatized hy-
drolysate established the 2S,3R configuration of the â-amino
acid unit in 1.
Next, we turned our attention to the configuration of the
Ibu units in 1-4. Synthetic standards of the reduced
Ibu unit, 4-amino-2,2-dimethyl-3-hydroxypentanoic acid
(Adhpa), were prepared as shown in Scheme 2. The
â-ketoester (18), prepared by the 1,1′-carbonyldiimidazole
activated coupling of N-Boc-L-Ala to the enolate of benzyl
acetate, was dimethylated with NaH and MeI.9,28 The gem-
dimethyl adduct (19) was isolated in 17% yield by silica
chromatography and eluted after the major product (53%),
(4S)-N-Boc-3-oxo-2,2,4-trimethyl-γ-lactam (20). Reduction
of 19 with NaBH4 provided a mixture of epimeric alcohols
(21),29 which were deprotected to afford a mixture of
(3R,4S)- and (3S,4S)-22 (Adhpa). Derivatization with L- and
DL-FDLA and HPLC analyses of these mixtures established
the elution order of the standards.30
F igu r e 1. GC-MS fragmentations of the Amha unit (7) found in 1
and the synthetic MAP unit (6).
A series of 2D NMR and 1D TOCSY experiments were
largely unsuccessful due to the extensive signal broadening
and doubling in the 1H and 13C NMR spectra of the
apparently chromatographically homogeneous material.
Variable-temperature NMR experiments between 70 and
-20 °C in a variety of solvents with and without LiCl13
failed to alter the conformational ratio and ameliorate the
situation. These experiments did indicate that 1 contained
at least eight amino acids, based on four N-methylamide
and four secondary amide proton signals.14 Lyngbyastatin
3 (1) was likely a depsipeptide on the basis of the prominent
vibrations at 1729 and 1637 cm-1 in the thin-film IR
spectrum that indicated ester and amide functionalities,
respectively.
These physical characteristics suggested that 1 was
related to the known L. majuscula metabolite lyngbyasta-
1
tin 1 (2). While the IR, UV, and H NMR spectra of 1 and
2 were virtually identical, a comparison of the two samples
by reversed-phase HPLC15 indicated that 1 was more
lipophilic than 2. This difference in polarity was attributed
to an extra methylene (δC 20.7) in 1 as revealed by a DEPT
experiment in CD3CN at 70 °C and confirmed by high-
resolution mass spectrometry.
Analysis of the derivatized acid hydrolysate16 of 1 by
chiral GC-MS17 established the location of the extra
methylene. The GC-MS chromatogram of this mixture did
not show a peak corresponding to the derivatized (2S,3R)-
3-amino-2-methylpentanoic acid unit (6, m/z 319) found in
2. A synthetic standard18 of this MAP unit exhibited two
predominant fragmentation pathways (Figure 1). In the
first pathway, successive homolytic cleavage of the aliphatic
side chain adjacent to the amide bond (9), McLafferty
rearrangement (12), and decarboxylation (14) generated
fragment ion peaks at m/z 290, 248, and 204, respectively.
In the second pathway, cleavage around the ester linkage
in 6 produced ion peaks at m/z 260 (8) and 204 (11)19 where
NaBH4 reduction31 of the natural products produced
derivatives that could be degraded to liberate Adhpa (22),

1358 J ournal of Natural Products, 2003, Vol. 66, No. 10
Sch em e 2. Synthesis of Adhpa Standardsa
Williams et al.
deuterium incorporation suggests very little enolization
occurs during the reduction and that (S)-Ibu-3 is present
in the original sample.
These results indicate that our cyanobacterial samples
of dolastatin 12 and lyngbyastatin 1 and 3 are mixtures of
two Ibu epimers. The configuration of the major Ibu epimer
in each of the three mixtures is R. The relative amounts
of the two epimers, as determined by HPLC analysis after
borohydride reduction, was 4:1 for both lyngbyastatin 1 (2)
and dolastatin 12 (3), but 2:1 for lyngbyastatin 3 (1). The
samples of 2 and 3 had both been isolated from the same
collection of L. majuscula, but the sample of lyngbyastatin
3 (1) had been obtained from several different collections
of L. majuscula from Apra Harbor.34 Since the collections
were subjected to slightly different isolation procedures,
this variation in the ratio of 1 might be due to some
epimerization during the isolation. The plausibility of this
event is supported by the molecular modeling of lyngby-
astatin 1 (2) and Ibu-epi-2 (data not shown), which suggests
that the S configuration for the Ibu unit is thermodynami-
cally favored.10
a
(i) 2 equiv of NaH, 18 equiv of Mel; (ii) NaBH4; (iii) H2, Pd/C; (iv) TFA.
Sch em e 3. Outcome of the NaBH4 Reductions of 1-4
The broadness in the NMR spectra of the cyanobacterial
samples of 1, 2, and 3 is most likely due to a combination
of factors. The presence of a minor diastereomer obviously
broadens the spectra considerably, but it has also been
suggested that the R- and S-Ibu diastereomers probably
have appreciably different amounts of cis and trans con-
formers around the (Ibu)-(N-Me-Ala) bond. Also in the case
of the R-Ibu compounds, the overall shape of the cis and
trans conformers differs significantly.10 All of these factors
likely contribute to the unusual degree of broadness in the
NMR spectra.
It has been proposed that dolastatins 11 (4) and 12 (3)
arise in the sea hare from feeding on cyanobacteria.8 The
isolation of another dolastatin analogue, lyngbyastatin 3
(1), from a cyanobacterium further supports this proposal.
The stereochemical differences in the Ibu units of the
compounds isolated from cyanobacteria and sea hares may
be caused by epimerization of the R to the thermodynami-
cally favorable S-Ibu configuration that occurred either in
the digestive gut of the sea hare or perhaps during the
isolation of the sea hare metabolites.2 However, a simpler
explanation is that the cyanobacterium eaten by the sea
hare produces dolastatin 12 with only the (S)-Ibu unit,
analogous to what is found in majusuclamide C.7b
from which the configurations of the Ibu units could be
determined.32 The reductions of 1-3 afforded two products
apiece (23-28) after HPLC purification of each reaction
mixture, while the reduction of 4 produced a single adduct
(29) (Scheme 3). All of the reaction products were identified
by HR-MS, and the major reduction products of lyngby-
astatin 3 (23) and dolastatin 12 (27) were also character-
ized by NMR (Tables 2 and 3) to ensure that the gross
structures of the original cyanobacterial metabolites were
correct. In general, the 1H NMR spectra of the major
reduction products showed a methine doublet (J ) 6.4 Hz)
at approximately δH 3.35 coupled only to the alcohol proton
(δH 5.43) and appeared to be a single compound. Con-
versely, the minor reduction products each appeared to be
mixtures of two compounds, as the HSQC spectrum of each
sample showed two protons at approximately δH 3.3 that
correlated to two different carbons at approximately 80
ppm.
Acid hydrolysis of the reduction products of 1-4 and
comparison with the Adhpa synthetic standards (22) by
Marfey analysis established the configuration of the Ibu
units in the cyanobacterial metabolites. As shown in Table
1, the reduction of the samples of 1-3 produced adducts
that contained Adhpa units in which the configuration of
C-4 was derived from both R- and S-Ibu, and in all three
cases, the minor products (24, 26, 28) had the configuration
of C-4 in Adhpa derived from S-Ibu. On the other hand,
the reduction of majusculamide C (4) produced a sample
(29) that contained an Adhpa unit derived from only S-Ibu.
It should be noted that only those samples that had broad
NMR spectra (1-3) produced reduction products corre-
sponding to both R- and S-Ibu units.
It is unlikely these mixtures are artifacts of the reduction
process. In a control experiment dolastatin 12 (3) was
reduced in 100% MeOH-d4 with NaBH4 and the purified
reaction products were analyzed by mass spectrometry. A
comparison of the minor reduction products from the
original and deuterium reduction experiments showed only
an 8% increase in the signal intensity of the M +1 peak
relative to the M+ ion in the latter.33 The magnitude of this
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a J asco-DIP-700 polarimeter at the sodium
D line (589 nm). UV spectra were taken on a Hewlett-Packard
8453 spectrophotometer. The IR spectra were recorded on a
Perkin-Elmer 1600 FTIR instrument as a film on a NaCl disk.
FABMS and HRFABMS were recorded in the positive mode
on a VG ZAB2SE spectrometer and MALDI on a DE-STR. The
NMR spectra of 1, 2, and 3 were recorded in CD3CN at 70 °C
on a Varian Unity Inova 400wb operating at 400 and 100 MHz
using the residual solvent signals as the internal reference,
while the NMR of the reduction products were performed at
500 and 125 MHz at 15 °C on a Varian instrument. NMR
analyses of the synthetic products were carried out at 300 MHz
using a Varian spectrometer. HPLC separations were per-
formed on a Beckman 110B apparatus coupled to an Applied
Biosystems 759A absorbance detector. All synthetic yields are
unoptimized. The following reagents were purchased from
Sigma-Aldrich: 1,5-difluoro-2,4-dinitrobenzene, L- and DL-
leucinamide hydrochloride, (R)- and (S)-N-benzyl-N-methyl-
benzylamine. Ethyl trans-2-hexanoate was purchased from
Alfa Aesar.

Lyngbyastatin 3 from Lyngbya majuscula
J ournal of Natural Products, 2003, Vol. 66, No. 10 1359
Ta ble 1. Stereochemistry of the Cyanobacterial Metabolites
cyanobacterial sample
broad NMR spectra
yes
reduction product(s)
Ibu configuration determineda
lyngbyastatin 3 (1)
23 (major)
24 (minor)
25 (major)
26 (minor)
27 (major)
28 (minor)
29
R (major) ) lyngbyastatin 3
S (minor) ) Ibu-epilyngbyastatin 3
R (major) ) lyngbyastatin 1
S (minor) ) Ibu-epilyngbyastatin 1
R (major) ) Ibu-epidolastatin 12
S (minor) ) dolastatin 12
S
lyngbyastatin 1 (2)
dolastatin 12 (3)
yes
yes
no
majusculamide C (4)
a
This is the configuration at C-4 of Adhpa that was detected in the L-FDLA-derivatized hydrolysate of the reduction product.
Biologica l Ma ter ia l. The cyanobacteria were collected on
the Tokai Maru shipwreck in Apra Harbor, Guam. The
organisms were identified by V. J . Paul, and vouchers are
maintained in formalin at the Smithsonian Marine Station,
Fort Pierce, FL.
Extr a ction . The freeze-dried cyanobacterium (95.1 g) des-
ignated NIH199 was sequentially extracted with a 1:1 mixture
of MeOH-CH2Cl2 and 30% aqueous ethanol to afford 1.29 and
4.60 g of lipophilic and aqueous extracts, respectively. NIH154
(72.4 g) and NIH143 (45.4 g) were treated in a similar manner
to provide 12.4 and 5.4 g of the lipophilic extracts, respectively.
The 30% aqueous EtOH extracts of NIH154 and NIH143 were
not cytotoxic and therefore not fractionated.
Isola tion of Lyn gbya sta tin 3 (1). Each extract was first
partioned between hexane and 80% aqueous MeOH. The
material in the polar layer was then subjected to a second
partitioning with n-BuOH and water. The organic residue was
separated on silica gel with CH2Cl2 and increasing amounts
of i-PrOH. The biologically active fractions (5 and 8%) were
separated on a prepacked Alltech C18 column (2000 mg) with
increasing amounts of CH3CN in H2O. Final purification of
the active fractions by RP-HPLC [Ultracarb ODS 30, 250 ×
10 mm, 70% aqueous CH3CN, flow rate 3 mL/min, detection
at 220 nm] afforded pure 1 (tR 18.5 min). NIH199 yielded 10.2
and 15.5 mg of 1 from the lipophilic and aqueous extracts,
respectively, while NIH154 and NIH143 gave 49.1 and 48.8
mg of 1 from the extracts, respectively.
Extr a ction a n d Isola tion of Lyn gbya sta tin 1 (2) a n d
Dola sta tin 12 (3). A 121.9 g sample of freeze-dried Lyngbya
majuscula NIH 198 was extracted with a 1:1 mixture of ethyl
acetate and methanol to give 2.5 g of lipophilic extract. The
remaining cell mass was subsequently extracted with a 3:7
mixture of ethanol and water to afford 13.5 g of aqueous
extract. Solvent partitioning of these two extracts as described
for 1 and subsequent Si chromatography with increasing
amount of i-PrOH in CH2Cl2 yielded two active fractions (5%
and 8% i-PrOH). Final purification of these fractions [Ultra-
carb ODS 30, 250 × 10 mm, flow rate 3 mL/min, detection at
220 nm] was achieved using an isocratic system of 55%
aqueous acetonitrile to afford samples of lyngbyastatin 1 (2)
(tR 40.4 min, 10.3 mg) and dolastatin 12 (3) (tR 46.7 min, 20.1
mg) from the lipophilic extract. The aqueous extract was
purified in the same manner to afford 3.6 mg of 2 and 6.8 mg
of 3.
Ch ir a l GC-MS An a lysis of Lyn gbya sta tin 3 (1). Lyng-
byastatin 3 (1) (1.0 mg) was dissolved in 6 N HCl (0.3 mL)
and heated to 118 °C for 18 h. The acid was removed under
N2, and the dry hydrolysate was treated at 100 °C for 45 min
with a mixture of 0.3 mL of 2-propanol and 50 µL of acetyl
chloride. The excess reagent was removed under N2 and the
residue treated at 100 °C for 15 min with 50 µL of a 1:1
solution of (CF3CF2CO)2O/CH2Cl2. After the mixture had cooled
to room temperature, the excess reagent was removed under
a stream of N2 and the resulting mixture of isopropyl esters
and N-(pentafluoropropionyl) amino acids was dissolved in 50
µL of MeOH and analyzed by GC-MS [Chirasil-Val column,
25 µm × 0.25 m, Alltech; 12 psi initial pressure, temperature
gradient of 40 to 120 °C with a heating rate of 3 °C/min].
(2S,3R)-3-Amino-2-methylpentanoic acid (6) had a tR of 18.6
min: m/z (intensity) 319 (5), 290 (25), 260 (42), 248 (40), 230
(50), 204 (100). Lyngbyastatin 3 (1) showed 3-amino-2-meth-
ylhexanoic acid (7) with a tR of 21.0 min: m/z (intensity) 333
(2), 290 (20), 274 (45), 248 (38), 230 (40), 218 (85), 204 (70),
176 (80).
Syn th esis of 3-Am in o-2-m eth ylh exa n oic Acid Sta n -
d a r d s: (3R,7R)-Eth yl-N-(r-m eth ylben zyl ben zyl)-3-a m i-
n oh exa n oa t e (15). A solution of (R)-(+)-N-benzyl-N-R-
methylbenzylamine (3.2 mmol) in THF (10 mL) was cooled to
-78 °C prior to the slow addition of n-BuLi (3.0 mmol). The
resulting pink solution was stirred for 30 min before the slow
addition of ethyl trans-2-hexenoate (2 mmol) in 2 mL of THF.
After stirring for 2 h at -78 °C, the reaction was quenched
with saturated aqueous NH4Cl. The solution was warmed to
room temperature and partitioned between diethyl ether and
water. Chromatography of the organic residue on silica with
petroleum ether (bp 40-60 °C) and diethyl ether (20:1)
resulted in 504 mg (50%) of 15: 1H NMR of 15 (300 MHz,
CDCl3) δH (integration, multiplicity; J in Hz) 7.33 (10, m), 3.91
(3, m), 3.86 (2, br s), 3.28 (1, m), 1.97 (2, d; 5.1), 1.54 (2, m),
1.25 (2, m), 1.09 (3, d; 7.1), 1.09 (3, t; 7.1), 0.88 (3, t; 7.2).
(2SR,3R,7R)-Eth yl-N-(r-m eth ylben zyl ben zyl)-3-am in o-
2-m eth ylh exa n oa te (16). A solution of 15 (0.3 mmol) in 2
mL of THF was added dropwise to a solution of KHMDS (0.5
mmol) in 10 mL of THF. The reaction mixture was stirred for
1 h before the addition of neat iodomethane (5 mmol). The
mixture was warmed to room temperature over 16 h and then
partitioned between diethyl ether and water to afford the crude
product (52 mg, 50%) as a 3:1 anti:syn mixture of C-2
diastereomers. The resulting mixture was used without further
purification. 1H NMR of the (2R,3R)-diastereomer as deter-
mined by 1D TOCSY experiments (300 MHz, CDCl3): δH
(position, multiplicity; J in Hz) 7.25 (Ar, m), 4.01 (OCH2 and
NCH, m), 3.87 and 3.74 (PhCH2, AB system;14.6), 3.13 (NCH,
m), 2.51 (MeCH, p; 7.0), 1.54 (NCCH2, m), 1.38 (MeCH2, m),
1.31 (NCHCH3, d; 6.0), 1.19 (OCH2CH3, t; 6.2), 0.97 (CHCH3,
d; 7.0), 0.89 (CH2CH3, t; 6.7). 1H NMR of the (2S,3R)-
diastereomer as determined by 1D TOCSY experiments (300
MHz, CDCl3): δH (position, multiplicity; J in Hz) 7.25 (Ar, m),
4.01 (OCH2 and NCH, m), 3.87 and 3.74 (PhCH2, AB system;
14.6), 2.84 (NCH, m), 2.48 (MeCH, p; 7.3), 1.54 (NCCH2, m),
1.32 (MeCH2, m), 1.31 (NCHCH3, d; 6.0), 1.19 (OCH2CH3, t;
6.2), 0.95 (CHCH3, d; 7.0), 0.85 (CH2CH3, t; 6.7).
The mixture of lyngbyastatin 3 (R-Ibu-1) and Ibu-epilyng-
byastatin 3 (S-Ibu-1) was obtained as a colorless oil: [R]27
D
-62° (c 0.12, CHCl3); UV (MeOH) λmax (log ꢀ) 201 (4.23), 209
(3.98), 221 (3.87), 277 (3.45) nm; IR (film) νmax 3386, 1729,
1637, 1513, 1466, 1248 cm-1; FABMS m/z [M + H]+ 1014, [M
+ Na]+ 1036; HRFABMS m/z [M + H]+ 1013.6337 (calcd for
C
52H82N8O12 1013.6399, 5.8 mDa error).
The mixture of lyngbyastatin 1 (R-Ibu-2) and Ibu-epilyng-
byastatin 1 (S-Ibu-2) was obtained as a clear glassy oil: [R]27
D
-17° (c 0.3, MeOH); UV (MeOH) λmax (log ꢀ) 236 (3.15), 277
(2.80), 284 (2.75) nm; IR (film) νmax 3320, 1791, 1673, 1525,
1461, 1402, 1279 cm-1; HRFABMS m/z 1021.5965 (calcd for
C
51H82N8O12Na 1021.5944, 2.1 mDa error).
The mixture of dolastatin 12 (S-Ibu-3) and Ibu-epidolastatin
12 (R-Ibu-3) was obtained as a clear glassy oil: [R]27 -54° (c
D
(2SR,3R)-3-Am in o-2-m eth ylh exa n oic Acid Hyd r och lo-
r id e (17). To a solution of 16 and its C-2 epimer (25 mg total)
in 6 mL of glacial acetic acid was added 14 mg of 20% Pd/C.
After 16 h under approximately 5 atm of H2, the mixture was
0.7, MeOH); UV (MeOH) λmax (log ꢀ) 234 (2.70), 241 (3.00), 243
(3.66), 277 (2.24), 283 (2.24) nm; IR (film) νmax 3316, 1791,
1672, 1525, 1461, 1402, 1278 cm-1; HRFABMS m/z 991.5818
(calcd for C50H80N8O11Na 991.5839, 2.1 mDa error).

1360 J ournal of Natural Products, 2003, Vol. 66, No. 10
Ta ble 2. NMR Spectral Data for 23 in CDCl3
Williams et al.
unit
Amha
C/H no.
δ
H (J in Hz)
δC
1H-1H COSY
ΗΜΒC
1
2
3
173.6, s
43.5, d
52.5, d
2, 7, 48
2.82, m
3.89, m
7.68, d (7.9)
1.38, m
1.20, m
1.22, m
0.86, t (6.4)
1.25, d (7.2)
3, 7
2, 3-NH, 4
3
2
6
6
3-NH
4
24.2, t
3
2
5
6
7
8
19.9, t
13.7, q
14.9, q
170.9, s
54.6, d
14.2, q
32.2, q
178.0, s
46.2, s
81.3, d
N-Me-Ala
9, 10
11
9
5.14, q (6.9)
1.38, d (6.9)
3.09, s
10
9
10
11
12
13
14
14-OH
15
15-NH
16
17
18
19
20
21
9
Adhpa
11, 14, 17, 18
17, 18
16, 17, 18
3.36, d (6.4)
5.43, d (6.4)
4.24, dq (8.6, 6.7)
6.04, d (8.6)
1.04, d (6.7)
1.26, s
14-OH
14
15-NH, 16
15
15
44.6, d
16
21.2, q
26.3, q
22.8, q
168.5, s
60.8, d
34.8, t
1.27, s
N,O-diMe-Tyr
15, 20, 21
21, 29
4.78, dd (10.2, 4.5)
2.96, dd (-17.7, 10.2)
2.90, dd (-17.7, 4.5)
21
20
20
22
128.4, s
130.3, d
114.3, d
158.7, s
55.3, q
29.3, q
169.5, s
58.4, d
26.8, d
18.0, q
18.2, q
29.9, q
169.2, s
41.5, t
21
23/27
24/26
25
28
29
30
31
32
33
34
35
36
37
7.12, d (8.2)
6.81, d (8.2)
24/26
23/27
21, 23/27, 25
22, 24/26, 25
28
3.74, s
2.93, s
20
N-Me-Val
29, 31
33, 34
31, 33, 34
34
4.61, d (10.7)
2.16, m
0.62, d (6.7)
0.18, d (6.9)
2.87, s
32
31, 33, 34
32
32
33
31
Gly
35, 37
4.22, dd (-18.9, 7.2)
4.10, d (-18.9)
7.09, d (7.2)
37u, 37-NH
37d
37d
37-NH
38
39
N-Me-Leu
169.9, s
54.1, d
36.6, t
37, 39, 40
42, 43
5.32, dd (9.5, 4.5)
1.72, m
1.66, m
40
40
39, 41
39, 41
40
41
41
41
42
43
44
45
46
1.36, m
24.6, d
23.3, q
21.9, q
29.4, q
169.0, s
41.2, t
0.98, d (6.2)
0.88, d (7.3)
2.92, s
Gly
44, 46
46, 48
4.46, dd (-18.1, 7.1)
3.81, dd (-18.1, 1.7)
7.27, dd (7.1, 1.7)
46-NH
46-NH
46
46-NH
47
48
49
50
HMVA
168.9, s
77.7, d
37.0, d
30.5, t
5.15, d (4.3)
2.04, m
1.58, m
1.20, m
0.90, t (7.3)
0.95, d (6.2)
49
48, 50, 52
48
48
49
49
50
49
51
52
11.4, q
15.0, q
48
filtered through a pad of Celite and the solvent removed under
N2. Half of this sample was dissolved in 6 N HCl and refluxed
for 18 h at 118 °C to yield 5.1 mg of 17 (100%). 1H NMR of
(2R,3R)- and (2S,3R)-17 obtained by 1D TOCSY experiments
was identical to literature data.22
Red u ction of Na tu r a l P r od u cts: Dih yd r o-m a ju scu la -
m id e C (29). A solution of 2 mg of 4 in 0.2 mL of methanol
was added to 4 mg of NaBH4. After stirring 25 min, another 4
mg aliquot of NaBH4 was added and the solution stirred for
another 25 min before quenching with 1 N HCl. The mixture
was partitioned between ethyl acetate and water. The organic
residue purified by C18 HPLC [Ultracarb ODS 30, 250 × 10
mm, 60% aqueous CH3CN, flow rate 3 mL/min, detection at
220 nm] afforded a single product (29, tR 39.5 min) with no
sign of 4 (tR 50.2 min): 13C NMR (CDCl3) δC 176.7, 173.4, 172.4,
170.50, 170.45, 170.0, 169.8, 169.7, 167.7, 158.6, 130.4, 128.7,
114.2, 80.3,35 77.9, 61.4, 61.0, 58.0, 55.3, 50.2, 47.5, 45.2, 44.4,
42.3, 41.3, 40.5, 37.5, 35.2, 29.9, 29.5, 29.3, 27.1, 26.8, 25.6,
24.7, 23.6, 22.4, 18.33, 18.26, 15.48, 15.44, 14.6, 13.8, 11.7, 10.9,
10.7, 8.9; HR-MALDI m/z 1009.5991 [calcd for C50H82N8O12
-
Na 1009.5944, 4.7 mDa error].
Dih yd r o-lyn gbya sta tin 3 (23, 24). A sample of 1 (6 mg)
was reduced as described for 4 and purified by HPLC (62%
aqueous CH3CN) to afford the minor (24, tR 31.3 min) and the
major reduction products (23, tR 37.9 min) in a ratio of 1:2.
1
23: H and 13C NMR, see Table 2; HR-MALDI m/z 1037.6310

Lyngbyastatin 3 from Lyngbya majuscula
J ournal of Natural Products, 2003, Vol. 66, No. 10 1361
Ta ble 3. NMR Spectral Data for 27 in CDCl3
unit
Map
C/H no.
δ
H (J in Hz)
δC
1H-1H COSY
HMBC
2, 3, 6
1
2
3
173.6, s
42.5, d
54.4, d
2.89, m
3.74, m
3, 6
2, 3-NH, 4
6
2, 4, 5
3-NH
4
7.73, d (7.1)
1.66, m
3
21.6, t
3, 4u, 5
2
1.18, m
3, 4d, 5
5
6
7
8
0.89, t (6.6)
1.25, d (8.2)
11.5, q
14.9, q
171.1, s
54.5, d
14.2, q
32.2, q
178.0, s
46.2, s
81.1, d
4
2
4
2
N-Me-Ala
3, 8, 9
9, 10
5.18, q (6.9)
1.38, d (6.9)
3.08, s
9
8
9
10
11
12
13
13-OH
14
14-NH
15
16
17
18
19
20
Adhpa
10, 13, 16, 17
13, 16, 17
15, 16, 17
3.35, d (5.6)
5.44, d (5.6)
4.23, dq (8.1, 6.7)
6.05, d (8.1)
1.04, d (6.7)
1.24, s
13-OH
13
14-NH, 15
14
14
44.6, d
15
14
21.3, q
26.1, q
22.7, q
168.4, s
60.8, d
34.8, t
1.27, s
N-Me-Phe
14, 19
20, 27
19
4.85, dd (10.4, 4.4)
3.05, dd (-14.3, 4.4)
2.97, dd (-14.3, 10.4)
20
19
19
21
136.5, s
129.3, d
128.9, d
127.1, d
29.3, q
169.5, s
58.4, d
26.8, d
18.0, q
18.1, q
29.0, q
169.2, s
41.2, t
20, 23/25
22/26
23/25
24
27
28
29
30
31
32
7.20, d (7.6)
7.26, t (7.6)
7.20, d (7.6)
2.92, s
23/25
22/26, 24
23/25
19
N-Me-Val
19, 27, 29
30, 31, 32
29, 31, 32
4.60, d (10.4)
2.12, m
0.60, d (6.7)
0.09, d (6.4)
2.86, s
30
29, 31, 32
30
30
33
34
35
29
33, 35
Gly
4.46, dd (-18.1, 7.1)
3.81, d (-18.1)
7.23, d (7.1)
35u
35d
35d
35-NH
36
37
N-Me-Leu
169.9, s
54.5, d
36.6, t
35, 37, 38
39, 42
37
5.32, dd (9.9, 4.2)
1.75, ddd (-14.6, 10.4, 4.2)
1.61, ddd (-14.6, 9.9, 4.0)
1.34, m
0.93, d (6.6)
0.86, d (6.4)
38
38
37, 38u, 39
37, 38d, 39
39
40
41
42
43
44
24.6, d
23.3, q
21.8, q
29.3, q
168.9, s
41.4, t
37, 38
38
38
2.91, s
Gly
37, 42, 44
4.19, d (-17.1)
3.99, dd (-17.1, 5.2)
7.06, d (5.2)
44u
44d, 44-NH
44u
44-NH
45
46
47
48
HMVA
169.1, s
77.8, d
36.9, d
24.2, t
44, 46
47, 48, 50
5.15, d (4.7)
2.04, m
1.49, m
47
46, 48, 50
47, 48u
48d
1.19, m
49
50
0.88, t (7.4)
0.92, d (6.9)
11.3, q
15.0, q
48d
47
48u
48d
[calcd for C52H86N8O12Na 1037.6257, 5.3 mDa error]. 24:36 HR-
MALDI m/z 1037.6273 [calcd for C52H86N8O12Na 1037.6257,
1.6 mDa error].
Dih yd r o-lyn gbya sta tin 1 (25, 26). A sample of 2 (2 mg)
was reduced as described for 4 and purified by HPLC (55%
CH3CN) to afford the minor (26, tR 37.7 min) and major
reduction products (25, tR 45.0 min) in a 1:4 ratio. 25:36 HR-
MALDI m/z 1023.6078 [calcd for C51H84N8O12Na 1023.6101,
2.3 mDa error]. 26:36 HR-MALDI m/z 1023.6057 [calcd for
MALDI m/z 993.5944 [calcd for C50H82N8O11Na 993.5995, 5.1
mDa error]. 28: 13C NMR (CDCl3) δC 178.0, 174.1, 170.7, 169.9,
169.1, 167.7, 137.3, 129.3, 128.9, 127.0, 80.9,35 78.0, 61.9, 58.8,
54.9, 46.4, 43.6, 41.4, 37.4, 36.7, 35.0, 32.7, 29.9, 29.6, 29.4,
27.1, 24.9, 24.3, 22.9, 22.1, 21.0, 18.6, 18.3, 15.8, 15.1, 14.6,
11.2; HR-MALDI m/z 993.6040 [calcd for
C50H82N8O11Na
993.5995, 4.5 mDa error].
Syn th esis of (3RS,4S)-4-Am in o-2,2-dim eth yl-3-h ydr oxy-
p en ta n oic Acid : (4S)-Ben zyl N-Boc-4-a m in o-3-oxo-p en -
ta n oa te (18). This reaction was carried out as previously
described,9 except the solution of LDA and benzyl acetate was
stirred for only 5 min before the addition of the acyl imidazole.
Purification as described9 afforded 18 in a yield of 50%. 1H
NMR (CDCl3) δH (integration, multiplicity; J in Hz): 7.38 (5,
C
51H84N8O12Na 1023.6101, 4.3 mDa error].
Dih yd r o-d ola sta tin 12 (27, 28). A sample of 3 (4 mg) was
reduced as described for 4 and purified by HPLC (55% CH3-
CN) to afford the minor (28, tR 44.4 min) and the major
reduction products (27, tR 55.9 min) in a 1:4 ratio. 27: 1H NMR,
13C NMR, 1H-1H COSY, and HMBC data, see Table 3; HR-

1362 J ournal of Natural Products, 2003, Vol. 66, No. 10
Williams et al.
s), 5.18 (2, s), 5.08 (1, d; 7.0), 4.36 (1, p; 7.0), 3.64 (1, d; -16.0),
3.57 (1, d; -16.0), 1.43 (9, s), 1.32 (3, d; 7.0).
with 0.01 M TFA over 50 min, flow rate 0.3 mL/min, detection
at 342 nm]. The mass spectra were collected in the negative
mode and had an ESI voltage of 4.6 kV with the auxiliary and
sheath gas pressure set at 5 units and 70 psi, respectively,
and the capillary heated to 200 °C. A mass range of m/z 438-
440 was covered. Analysis of the L-FDLA-derivatized hydroly-
sate gave two peaks at 32.3 and 35.8 min. The former was
identified as N-Me-L-Leu by co-injection of a derivatized
standard, and the latter therefore was the derivatized Amha
unit. Analysis of the DL-FDLA-derivatized hydrolysate gave
four peaks which eluted at 30.6, 32.3, 35.3, and 35.8 min,
which corresponded to (D-FDLA)-Amha, (D-FDLA)-(N-Me-L-
Leu), (L-FDLA)-(N-Me-L-Leu), and (L-FDLA)-Amha, respec-
tively. Since the D-FDLA-derivatized Amha unit eluted before
the L-FDLA-derivatized Amha unit, based on the proposed
separation mechanism of the FDLA derivatives, the absolute
configuration of the Amha unit at C-3 was R.23 The retention
times of L-FDLA-derivatized N-Me-DL-Leu were 32.3 and 35.3
min for the L- and D-derivatized amino acid, respectively.
(4S)-Ben zyl N-Boc-4-a m in o-2,2-d im eth yl-3-oxop en ta n -
oa te (19). To a solution of 150 mg of 18 and 9 equiv of MeI in
THF (5 mL) was added 1 equiv of NaH (65% dispersion). The
solution was stirred for 1 h before the addition of one more
equivalent of NaH and nine more equivalents of MeI. After
16 h, 100 mL of diethyl ether was added and the solution was
sequentially partitioned with sodium thiosulfate and brine.
The organic layer was dried over MgSO4 and the solvent
removed in vacuo. Purification over silica with 6:1 hexane-
EtOAc yielded 60 mg (53%) of (4S)-N-Boc-2,2,4-trimethyl-3-
oxo-γ-lactam first (20), followed by 33 mg of 19 (17%). 1H NMR
of 19 (CDCl3): δH (integration, multiplicity; J in Hz) 7.33 (5,
m), 5.15 (2, s), 4.89 (1, d; 7.9), 4.63 (1, p; 7.9), 1.59 (3, s), 1.44
(3, s), 1.41 (9, s), 1.18 (3, d; 7.9). Lactam 20: 1H NMR (CDCl3)
δH (integration, multiplicity; J in Hz) 4.35 (1, q; 6.9), 1.56 (9,
s), 1.52 (3, d; 7.0), 1.30 (3, s), 1.28 (3, s).
(3SR,4S)-4-Am in o-2,2-d im et h yl-3-h yd r oxyp en t a n oic
Acid (22). A solution of 19 (33 mg) in methanol was added to
4 equiv of NaBH4 at 0 °C. The mixture was stirred for 30 min,
until it was negative to 2,4-dinitrophenylhydrazine, and then
it was quenched with 1 N HCl. It was subsequently partitioned
between EtOAc and brine, then concentrated to dryness to
yield 20 mg (62%) of (3SR,4S)-benzyl N-Boc-4-amino-2,2-
dimethyl-3-hydroxypentanoate (21). The benzyl group was
removed by hydrogenation over 30% Pd/C in methanol at room
temperature for 12 h. The suspension was filtrated through
Celite and evaporated to dryness. The residue was dissolved
in concentrated TFA and stirred for 15 min before removing
the acid under a stream of nitrogen to afford 6.3 mg (69%) of
Ad va n ced Ma r fey: â-Am in o Acid s. A portion of the
sample (25 µL) prepared for the LC-MS analysis of the â-amino
acids was analyzed by RP-HPLC [Bondclone 10 C18, 300 × 7.8
mm, a linear gradient of 20 to 80% CH3CN with 0.01 M TFA
over 50 min, flow rate 3 mL/min, PDA detection]. The retention
times of the L-FDLA-derivatized amino acids in the hydroly-
sate were Gly (21.5 min), N-Me-L-Ala (22.1 min), N-Me-L-Val
(25.8 min), N,O-diMe-L-Tyr (26.5 min), N-Me-L-Leu (28.2 min),
and (2S,3R)-17 (31.2 min). The retention times of the D-FDLA-
derivatized amino acids in the DL-FDLA-derivatized hydroly-
sate were Gly (21.5 min), N-Me-L-Ala (22.9 min), (2S,3R)-17
(26.5 min), N-Me-L-Val (29.2 min), N,O-diMe-L-Tyr (30.3 min),
and N-Me-L-Leu (30.7 min). The retention times of L-FDLA-
derivatized standards were (2R,3R)-17 (30.3 min) and (2S,3R)-
17 (31.3 min), while the D-FDLA-derivatized standards were
detected at (2S,3R)-17 (26.7 min) and (2R,3R)-17 (27.2 min).
1
22. The H NMR of the major diastereomer in the mixture of
22: (MeOH-d4) δH (integration, multiplicity; J in Hz) 3.85 (1,
d; 1.8), 3.51 (1, qd; 6.0, 1.8), 1.25 (3, s),37 1.24 (3, d; 6.0),37 1.18
(3, s). Minor diastereomer of 22: δH (MeOH-d4) 3.67 (0.7, d;
5.6), 3.39 (0.7, qd; 6.5, 5.6), 1.30 (2, s), 1.27 (2, s), 1.24 (2, d;
6.5).37
Ma r fey An a lysis of th e Red u ction P r od u cts 23-29. The
samples were hydrolyzed for 24 h as described for 1 and
derivatized with L-FDLA before HPLC analyses [YMC-AQ
ODS, 250 × 10 mm, a linear gradient of 40 to 50% aqueous
CH3CN with 0.1% TFA (pH 4.13) over 60 min, flow rate 2.5
mL/min, PDA detection]. The retention time of L-FDLA-
(3SR,4S)-22 was 20.3 min, while D-FDLA-(3SR,4S)-22 eluted
at 22.6 min. A peak corresponding to L-FDLA-(3SR,4S)-22
(20.3) was found in 24, 26, 28, and 29, which was confirmed
by co-injection of the appropriate standard and by comparison
of the UV spectrum. The stereochemistry of Ibu units in the
major reduction products 23, 25, and 27 was determined by
LC-MS due to overlap in the HPLC trace.
Absolu te Ster eoch em istr y of th e Am in o Acid -Der ived
Un its: Ch ir a l HP LC. The hydrolysate of 1 was analyzed by
chiral HPLC, and the retention times were compared with
authentic standards [Column Chirex Phase 3126 (D), 250 ×
4.6 mm, Phenomenex, solvent 2 mM CuSO4-MeCN (95:5) for
Gly, N-Me-Ala, N-Me-Val, and N-Me-Leu; N,O-diMe-Tyr with
2 mM CuSO4-MeCN (90:10), flow rate 0.8 mL/min, detection
at 254 nm, except 2-hydroxy-3-methylvaleric acid, which was
determined on a CHIRALPAK MA(+), 50 × 4.6 mm, Diacel
Chemical Industries, Ltd., 2 mM CuSO4-MeCN (85:15), flow
rate 0.8 mL/min, detection at 254 nm]. The retention times of
the standards were Gly (7.1 min), N-Me-L-Ala (9.0 min), N-Me-
D-Ala (9.8 min), N-Me-L-Val (14.1 min), N-Me-D-Val (19.2 min),
N-Me-L-Leu (52.1 min), N-Me-D-Leu (79.0 min), N,O-diMe-L-
Tyr (81.5 min), N,O-diMe-D-Tyr (87.1 min), D-2-hydroxy-3-
methylvaleric acid (40.1 min), D-allo-2-hydroxy-3-methylvaleric
acid (34.1 min), L-allo-2-hydroxy-3-methylvaleric acid (52.0
min), and L-2-hydroxy-3-methylvaleric acid (66.2 min). The
retention times of the amino acid components of the hydroly-
sate were Gly (7.1 min), N-Me-L-Ala (9.0 min), N-Me-L-Val
(14.1 min), N-Me-L-Leu (52.1 min), L-2-hydroxy-3-methylva-
leric acid (66.2 min), and N,O-diMe-L-Tyr (81.5 min). The
identities of the peaks were confirmed by co-injection.
LC-MS An a lysis of â-Am in o Acid s. Before the synthetic
standards of the Amha unit were prepared, the configuration
at C-3 of the Amha unit in 1 was determined by LC-MS. The
sample of lyngbyastatin 3 (1) (1 mg) was hydrolyzed in 6 N
HCl at 118 °C for 16 h, after which the sample was dried under
N2. The residue was twice dissolved in 25% aqueous triethyl-
amine and evaporated. The hydrolysate was resuspended in
50 µL of water, and then 20 µL of 1 M NaHCO3 and 50 µL of
1% FDLA (either the L- or DL-mixture as required) were added.
The reaction was maintained at 80 °C for 3 min until the
yellow solution turned orange. After cooling to room temper-
ature 20 µL of 1 N HCl was added and the yellow solution
was diluted with 0.8 mL of CH3CN. The separations of the L-
and DL-FDLA derivatives were performed on a Hydrobond-
ODS [100 × 3.0 mm, a linear gradient of 20 to 80% CH3CN
LC-MS An a lyses of th e Red u ction P r od u cts. The sepa-
rations of the L- and DL-FDLA derivatives were performed on
a Hydrobond-ODS [100 × 3 mm, a linear gradient of 40 to 50%
CH3CN with 0.25% acetic acid over 60 min, flow rate 0.3 mL/
min, detection at 342 nm]. The mass spectra were collected in
the positive mode and had an ESI voltage of 4.6 kV with the
auxiliary and sheath gas pressure set at 5 units and 70 psi,
respectively, and the capillary heated to 200 °C. A mass range
of m/z 455.4-456.4 was covered. The retention time of L-FDLA-
(3SR,4S)-22 was 18.2 min. The DL-FDLA-22 sample gave three
peaks (4:3:1 ratio) at 18.2, 20.0, and 24.8 min. The presence
of three peaks indicated that D-FDLA-(3R,4S)-22 and D-FDLA-
(3S,4S)-22 were resolved under the LC-MS conditions. The
elution order of these last two peaks was not conclusively
established, since it was irrelevant to the analysis, but (3R,4S)-
22 is likely the major reduction product with a retention time
of 20.0 min.29 The L-FDLA-derivatized acid hydrolysate of the
major reduction products 23, 25, and 27 contained a peak at
20.0 min with the correct mass for D-FDLA-22 indicating an
R configuration in the Ibu units of the natural products. The
L-FDLA-derivatized acid hydrolysate of the minor reduction
products (24, 26) of lyngbyastatins 3 and 1 were also analyzed
by LC-MS and provided peaks of the correct mass that coeluted
with L-FDLA-(3SR,4S)-22 (18.2), confirming the S configura-
tion in these natural products.

Lyngbyastatin 3 from Lyngbya majuscula
J ournal of Natural Products, 2003, Vol. 66, No. 10 1363
(19) The derivatives 6 and 7 can be converted to 11 and 13, respectively,
in one step by â-scission adjacent to the amide linkage. This one-
step pathway undoubtedly competes with the two- and three-step
processes shown in Figure 1. We thank the reviewer who brought
this to our attention.
(20) Horgen, F. D.; Yoshida, W. Y.; Scheuer, P. J . J . Nat. Prod. 2000, 63,
461-467.
(21) Luesch, H.; Williams, P. G.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J .
J . Nat. Prod. 2002, 65, 996-1000.
Ack n ow led gm en t. We thank the National Cancer Insti-
tute for providing support through R01 grant CA12623 and
NCNPDDG grant CA53001. The upgrades to the spectrom-
eters used in this research were funded by grants from the
CRIF Program of the National Science Foundation (CHE-
9974921), the Air Force Office of Scientific Research (F49620-
01-1-0524), and the Elise Pardee Foundation. The cyanobac-
teria were collected by D. Ginsburg. The high-resolution mass
spectral analyses were carried out by the UCR Mass Spec-
trometry Facility, Department of Chemistry, University of
California, and LC-MS analyses were provided by A. Franke
and L. Cluster at the University of Hawaii, Cancer Research
Institute. We thank G. Tien and M. Lieberman for the
bioassays, W. Y. Yoshida and W. Niemczura for the NMR
spectra, and M. Burger for the GC-MS data. We also thank
H. Luesch for his suggestions and comments.
(22) Kimura, J .; Takada, Y.; Inayoshi, T.; Nakao, Y.; Gilles, G.; Yoshida,
W. Y.; Scheuer, P. J . J . Org. Chem. 2002, 67, 1760-1767.
(23) (a) Fujii, K.; Yoshitomo, I.; Oka, H.; Suzuki, M.; Harada, K. Anal.
Chem. 1997, 69, 5146-5151. (b) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka,
H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 3346-3352.
(24) Davies, S. G.; Walters, I. A. S. J . Chem. Soc., Perkin Trans. 1 1994,
9, 1129-1139.
(25) The L- and DL-FDLA derivatizing reagents were prepared from L- and
DL-leucinamide, respectively, according to: Marfey, P. Carlsberg Res.
Commun. 1984, 49, 591-596.
(26) It should be noted that our proposed elution order [ent-(2R,3S), ent-
(2S,3S), (2R,3R), (2S,3R)] of the DL-FDLA derivatives differs from
that reported for the L-(1-fluoro-2,4-dinitrophenyl)-5-alaninamide (L-
FDAA) derivatives [(2S,3S), (2R,3S)].22 Apparently the side chain of
the amino amide has some effect on the elution order. Our elution
order was assigned based on the following: (1) The major peak in
the L-FDLA-derivatized mixture of 17 was assigned as (2R,3R) and
the minor (2S,3R) as expected from synthesis. (2) The DL-FDLA-
derivatized mixture of 17 gave four peaks in a ratio of 1:3:3:1 that
were assigned as (ent-2R,3S), (ent-2S,3S), (2R,3R), and (2S,3R),
respectively, as expected from synthesis. (3) The DL-FDLA-derivatized
hydrolysate of 1 contained peaks that coeluted with the first- and
last-eluting derivatized standards. This indicated that the first- and
last-eluting peaks when underivatized were enantiomers. (4) During
our structure elucidation of ulongamides A-C,21 which contain
(2R,3R)-Amha, the second- and third-eluting standards were present
in the DL-hydrolysate. Thus the underivatized second- and third-
eluting standards were enantiomers.
Su p p or tin g In for m a tion Ava ila ble: 2D NMR spectra of 23, 27;
1H and 13C NMR spectra of 1, 2, 3, 29; 1H NMR spectra of 24, 25, 26,
28. This material is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces a n d Notes
(1) Yamada, K.; Kigoshi, H. Bull. Chem. Soc. J pn. 1997, 70, 1479-1489,
and references therein.
(2) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd, M.
R.; Boettner, F. E.; Doubek, D. L.; Schmidt, J . M.; Chapuis, J .-C.
Tetrahedron 1993, 49, 9151-9170.
(3) Luesch, H.; Moore, R. E.; Paul, V. J .; Mooberry, S. L.; Corbett, T. H.
J . Nat. Prod. 2001, 64, 907-910.
(4) Mitchell, S. S.; Faulkner, D. J .; Rubins, K.; Bushman, F. D. J . Nat.
Prod. 2000, 63, 279-282.
(27) The same elution order with respect to C-3 (3S elutes before 3R) was
seen when synthetic MAP units were derivatized with the com-
mercially available Marfey reagent (L-FDAA). In this case the C-2
diastereomers could not be separated though. See: Williams, D. E.;
Burgoyne, D. L.; Rettig, J . J .; Andersen, R. J .; Faith-Afshar, Z. R.;
Allen, T. M. J . Nat. Prod. 1993, 56, 545-551.
(5) 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.
(6) Nogle, L.; Gerwick, W. H. J . Nat. Prod. 2002, 65, 21-24.
(7) (a) Lyngbyastatin 2 & Norlyngbyastatin 2: Luesch, H.; Yoshida, W.
Y.; Moore, R. E.; Paul, V. J . J . Nat. Prod. 1999, 62, 1702-1706. (b)
Symplostatin 1: Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore,
R. E.; Nagle, D. G.; Paul, V. J .; Mooberry, S. L.; Corbett, T. H.;
Valeriote, F. A. J . Nat. Prod. 1998, 61, 1065-1077. (c) Symplostatin
2: Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle,
D. G.; Paul, V. J . J . Nat. Prod. 1999, 62, 655-658. (d) Majusculamide
C: Carter, D. C.; Moore, R. E.; Mynderse, J . S.; Niemczura, W. P.;
Todd, J . S. J . Org. Chem. 1984, 49, 236-241. (e) 57-normajusculamide
C: Mynderse, J . S.; Hunt, A. H.; Moore, R. E. J . Nat. Prod. 1988, 51,
1299-1301.
(28) HPLC analysis of the L-FDLA derivative in the final product (22)
indicated approximately 15% epimerization.
(29) NaBH4 reductions on related amino acid-derived ketoesters generally
occur via a chelation-controlled mechanism to provide the 3R,4S
diastereomer as the major product. See: (a) Maibaum, J .; Rich, D.
H. J . Org. Chem. 1988, 53, 869-873. (b) Schuda, P. J .; Greenlee, W.
J .; Chakravarty, P. K.; Eskola, P. J . Org. Chem. 1988, 53, 873-875.
(30) Under the HPLC analysis conditions, the C-3 diastereomers of 22
were not resolved; that is, only a single peak was detected when the
mixture of L-FDLA-derivatized (3R,4S)- and (3S,4S)-22 was injected.
Likewise, when the D-FDLA-derivatized mixture was analyzed on the
Bondclone C18 column the standards equivalent to (3S,4R)- and
(3R,4S)-22 appeared as a single peak, but when these samples were
(8) 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.
(9) Bates, R. B.; Brusoe, K. G.; Burns, J . J .; Caldera, S.; Cui, W.;
Gangwar, S.; Gramme, M. R.; McClure, K. J .; Rouen, G. P.; Schadow,
H.; Stessman, C. C.; Taylor, S. R.; Vu, V. H.; Yarick, G. V.; Zhang,
J .; Pettit, G. R.; Bontems, R. J . Am. Chem. Soc. 1997, 119, 2111-
2113.
(10) Bai, R.; Bates, R. B.; Hamel, E.; Moore, R. E.; Nakkiew, P.; Pettit, G.
R.; Sufi, B. A. J . Nat. Prod. 2002, 65, 1824-1829.
(11) Geitler, L. Cyanophyceae. In Rabenhorst’s Kryptogamen-Flora; Aka-
demische Verlagsgesellschaft: Leipzig, 1932; Vol. 14.
submitted for LC-MS analysis on
a different C18 column, the
standards equivalent to (3S,4R)- and (3R,4S)-22 resolved into two
peaks.
(31) In general the stereochemical outcome (Cram vs anti-Cram) of the
reductions of the natural products was irrelevant to our analysis.
(32) We tried several of other strategies to determine the Ibu configuration
before settling on reducing the ketone. These include Baeyer-Villiger
oxidations of 1 or its methanolysis product with m-CPBA, peracetic
acid, trifluoroperacetic acid, or hydrogen peroxide,
a Beckmann
(12) 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. H. 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.
(13) The formation of complexes between inorganic salts and peptides can
sometimes alter the conformational ratio. Kofron, J . L.; Kuzmic, P.;
Kishoe, V.; Gemmecker, G.; Fesik, S. W.; Rich, D. H. J . Am. Chem.
Soc. 1992, 114, 2670-2675.
rearrangement of an oxime derivative of the methanolysis product
of 1, and reductive deoxygenation with tosylhydrazine and sodium
cyanoborohydride. If successful, the first two routes would have
allowed the configuration of the Ibu unit to be deduced by the presence
of L- or D-Ala after hydrolysis, while the third route would have
allowed direct application of the advanced Marfey technique without
synthetic standards.
(33) On average the ratio of the two signals was 61 and 53% for the minor
reduction products obtained in MeOH-d4 and MeOH, respectively.
The theoretical calculated ratio of (M + 1 + H)+/(M + H)+ is 61% for
C
50H82N8O11.
(14) The 1H NMR spectrum of 1 at -20 °C in CD3CN showed four methyl
singlets at approximately 2.8 ppm and four secondary amide proton
signals at approximately 7.9, 7.4, 7.5, and 6.9 ppm.
(15) Econosil C18, 250 × 10 mm, linear gradient 20% CH3CN in H2O to
100% CH3CN over 20 min then 100% for 40 min, flow rate 2 mL/
min, detection at 220 nm; 1 (tR 25.4 min), 2 (tR 24.5 min).
(16) The acid hydrolysate was derivatized to produce the isopropyl
N-(pentafluoropropionyl) amino esters.
(34) The samples of 1 from NIH199, -153, and -143 were combined before
the stereochemistry of the Ibu unit was known. This precluded
determining if each collection had the same ratio of epimers as might
be expected if the diastereomers were artifacts of the isolation
procedure and not due to biosynthetic differences in the strains.
(35) This signal is not present in the starting material.
(36) Too little material was isolated to characterize by 13C NMR spectrum.
(37) These three signals overlapped significantly and had a combined
integration of 8 relative to the other observed signals.
(17) Hartmut, F.; Nicholson, G. J .; Bayer, E. J . Chrom. Sci. 1977, 15, 174-
177.
(18) Provided by Professor Robert Bates at the University of Arizona.
NP0302145