Isolation and structure elucidation of the new fungal metabolite (-)-xylariamide A

Article abstract of DOI:10.1021/np050025h

Chemical investigations of the terrestrial microfungus Xylaria sp. have afforded the new natural product (-)-xylariamide A (1). The gross structure of 1 was determined by interpretation of 1D and 2D NMM, UV, IR, and MS data. Confirmation of the structure and the absolute stereochemistry of 1 were determined by the total synthesis of (4-)-xylariamide A (2). Synthetic 2 was produced by N,O-bis(trimethylsilyl)-acetamide-induced coupling of 3-chloro-L-tyrosine (3) with (E)-but-2-enedioic acid 2,5-dioxo-pyrrolidin-1-yl ester methyl ester (4). Optical rotation comparison of 1 with 2 indicated that the natural product (1) contained 3-chloro-D-tyrosine. Both enantiomers of xylariamide A were tested in a brine shrimp lethality assay, and only the natural product (1) showed toxicity.

Full text of DOI:10.1021/np050025h

J. Nat. Prod. 2005, 68, 769-772
769
Isolation and Structure Elucidation of the New Fungal Metabolite
(-)-Xylariamide A
Rohan A. Davis*
Chemical Biology Program, Eskitis Institute, Griffith University, Brisbane, QLD 4111, Australia
Received January 24, 2005
Chemical investigations of the terrestrial microfungus Xylaria sp. have afforded the new natural product
(-)-xylariamide A (1). The gross structure of 1 was determined by interpretation of 1D and 2D NMR,
UV, IR, and MS data. Confirmation of the structure and the absolute stereochemistry of 1 were determined
by the total synthesis of (+)-xylariamide A (2). Synthetic 2 was produced by N,O-bis(trimethylsilyl)-
acetamide-induced coupling of 3-chloro-L-tyrosine (3) with (E)-but-2-enedioic acid 2,5-dioxo-pyrrolidin-
1-yl ester methyl ester (4). Optical rotation comparison of 1 with 2 indicated that the natural product (1)
contained 3-chloro-D-tyrosine. Both enantiomers of xylariamide A were tested in a brine shrimp lethality
assay, and only the natural product (1) showed toxicity.
Several thousand organohalogen compounds have been
identified as biosynthetic products from living organisms.1
While the oceans are the single largest source of naturally
occurring organohalogens, many terrestrial plants, bacte-
ria, insects, and lichens have also been identified as
producers of halogenated natural products.2 Both marine-
and terrestrial-derived fungi produce chlorinated metabo-
lites; examples include pestalone,3 trichodermamide B,4
helicusins A-D,5 and lachnumon.6 Chlorinated fungal
metabolites containing the 3-chloro-4-hydroxyl phenyl
subunit are rare. To date only four such compounds have
been isolated;7 these include coniothyriomycin,8 3-chloro-
4-hydroxybenzeneethanol,9 3-chloro-4-hydroxyphenylacetic
acid,10 and 3-chloro-4-hydroxyphenylacetamide.11 We have
recently embarked on a research program looking for new
structural and bioactive metabolites from microfungi iso-
lated from Australian endemic plants.11,12 Examination of
a local rainforest tree, Glochidion ferdinandi (family Eu-
phorbiaceae), afforded several microfungal strains, one of
which was identified as Xylaria sp. (family Xylariaceace).
Chemical research on this particular strain has already
yielded two new xanthones12 and the novel metabolite
3-chloro-4-hydroxyphenylacetamide.11 Ongoing investiga-
tions of the EtOAc extract resulting from the fungal isolate
being cultured on solid media have resulted in the isolation
of a new chlorinated natural product. Herein we report the
isolation, structure elucidation, and absolute stereochem-
ical determination of the novel metabolite that we have
named (-)-xylariamide A (1).
1716 and 1667 cm-1, suggesting that 1 contained two or
more carbonyl groups. The 1H NMR spectrum of 1 con-
tained two exchangeable signals [δ 9.95, 8.80], five sp2
methine signals [δ 7.17, 7.05, 6.97, 6.84, 6.54], three mid-
field multiplets [δ 2.77, 3.00, 4.46], and a methoxyl singlet
[δ 3.71]. The 13C NMR spectrum of 1 displayed 14 signals,
of which 11 resonated between δ 119 and 173. The HSQC
spectrum enabled all the proton signals to be assigned to
their directly attached carbons. The three aromatic methine
signals at δ 7.17 (d, J ) 1.5 Hz, 1H), 6.84 (d, J ) 8.5 Hz,
1H), and 6.97 (d, J ) 8.5, 1.5 Hz, 1H) were assigned to a
1,3,4-trisubstituted benzene ring. This system was oxygen-
ated at C-4 of the aromatic ring on the basis of strong 3JCH
HMBC correlations from both H-2 (δ 7.17) and H-6 (δ 6.97)
to a carbon at δ 151.6. H-2 and H-6 also showed strong
HMBC correlations to a carbon at δ 35.5, to which the
diastereotopic protons δ 2.77 (H-7a) and 3.00 (H-7b) where
attached. ROESY correlation between H-7a and H-7b with
both methine protons H-2 and H-6 confirmed that C-7 (δ
35.5) was a benzylic carbon. The gCOSY spectrum and 1H-
1H coupling constants revealed that the diastereotopic
protons H-7a [δ 2.77 (dd, J ) 14.5, 10.0 Hz, 1H)] and H-7b
[δ 3.00 (dd, J ) 14.5, 5.0 Hz, 1H)] were positioned next to
the aliphatic methine proton H-8 [δ 4.46 (ddd, J ) 10.0,
8.0, 5.0 Hz, 1H)], which also showed a strong COSY
correlation to the exchangeable proton at 8-NH [δ 8.80 (d,
J ) 8.0 Hz, 1H)]. These characteristic data suggested that
(-)-xylariamide A contained a derivative of the aromatic
R-amino acid tyrosine. Strong HMBC correlations from
H-7a, H-7b, and H-8 to a carboxylic acid carbonyl at δ 172.3
provided further proof for this assignment. The olefinic
protons at δ 6.54 (dd, J ) 15.5 Hz, 1H) and 7.05 (d, J )
15.5 Hz, 1H) were assigned to an isolated E-double bond
on the basis of the multiplicity and magnitude of the
coupling constants.13 Both these protons showed HMBC
correlations to the carbonyl carbons at δ 162.7 and 165.4.
The latter carbon was assigned to a methyl ester moiety
on the basis of the only observed HMBC correlation from
the methoxyl singlet at δ 3.71 (3H) to C-13. Linkage of this
methyl ester terminated E-double bond to the amino acid
portion of the molecule was achieved via HMBC correla-
tions from both the olefinic proton (H-11) and amide proton
(8-NH) to the amide carbonyl at δ 162.7 (C-10). A strong
ROESY correlation between H-11 and 8-NH further sup-
ported this linkage. Although no 2D NMR correlations were
The fungus Xylaria sp. (FRR 5657) was grown on damp
white rice under static conditions, then extracted with
EtOAc. This extract was initially separated by C18 flash
column chromatography using H2O and increasing amounts
of CH3OH. An early eluting fraction was subjected to C18
preparative HPLC (CH3OH/H2O) followed by phenyl pre-
parative HPLC (CH3OH/aq TFA) to yield pure (-)-xylari-
amide A (1, 0.9 mg).
Compound 1 was isolated as an optically active pale
yellow gum and was assigned the molecular formula
C14H14NO6Cl on the basis of (-)-HRESIMS and 1H and 13
C
NMR spectral data. The (-)-LRESIMS isotopic pattern of
1 confirmed the presence of one chlorine atom.13 The IR
spectrum displayed two strong and broad absorptions at
* To whom correspondence should be addressed. Tel: +61 7 3875 6000.
Fax: +61 7 3875 6001. E-mail: r.davis@griffith.edu.au.
10.1021/np050025h CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/14/2005

770 Journal of Natural Products, 2005, Vol. 68, No. 5
Scheme 1. Total Synthesis of (+)-Xylariamide A (2)a
Notes
Figure 1. Structure of (-)-xylariamide A (1).
a (i) LiOH, acetone, rt, 1 h. (ii) N-Hydroxysuccinimide, EDCl, CH3CN,
rt, 24 h. (iii) 3-Chloro-L-tyrosine (3), BSA, DMF, 55 °C, 24 h.
Figure 2. Structure of cryptophycin A (7). Shared structural motif of
7 with 1 is outlined (- - -).
observed for the broad exchangeable signal at δ 9.95, this
was assigned to a hydroxyl group attached to C-4 of the
experiments performed on a 600 MHz Varian spectrometer
equipped with a triple resonance cold probe. These data
showed a JCH correlation from 1-OCH3 to C-2 in both
1
aromatic ring on the basis of the H chemical shift of this
hydroxyl moiety and the 13C chemical shift of this carbon
(δ 151.6). By default, the chlorine atom present in 1 was
attached to C-3 of the trisubstituted benzenoid system.
Hence a 3-chloro-4-hydroxyphenyl system was established.
The NMR data for this system showed only minor discrep-
4
compounds 4 and 5.
Interestingly, the natural product (-)-xylariamide A (1)
shares a structural motif with the cyanobacteria-derived
depsipeptide and potent antitumor agent cryptophycin A
(7)18 (Figure 2). Although compound 1 shared structural
homology with only a small portion of cryptophycin A (7),
this observation prompted us to test the natural product
(1) and synthetic enantiomer (2) for toxicity.
1
ancies (13C, <1.5 ppm; H, <0.09 ppm) with the aromatic
portions of the previously isolated fungal metabolites,
3-chloro-4-hydroxyphenylacetic acid and 3-chloro-4-hy-
droxyphenylacetamide.11 Although the carboxylic acid pro-
1
ton (9-OH) was not observed in the H NMR spectrum of
Both (-)-xylariamide A (1) and (+)-xylariamide A (2)
were screened for toxicity in a brine shrimp (Artemia
salina) lethality assay.19 The synthetic enantiomer (2)
showed no toxicity when screened at 20 or 200 µg/mL, while
the natural product (1) showed 0% and 71% lethality at
20 and 200 µg/mL, respectively. Unfortunately, lack of
material of 1 prevented us from calculating a LD50 value
in the brine shrimp lethality assay or testing (-)-xylari-
amide A in a panel of human carcinoma cell lines. The
synthesis of (-)-xylariamide A (1) is currently underway;
more material will allow further biological evaluations of
this new chlorinated fungal natural product to be per-
formed.
1, the (-)-LRESIMS data provided evidence of a carboxylic
acid group due to the fragment ions corresponding to
[M - 45]-.13 Hence the gross structure for 1 was assigned
to xylariamide A.
To determine the absolute stereochemistry of 1 and
obtain more material for future biological testing, we
synthesized xylariamide A using previously published
peptide chemistry.14 Regardless of which enantiomer of
xylariamide A we synthesized, the comparison of optical
rotation sign would enable us to unequivocally assign the
absolute stereochemistry of the natural product. The
synthetic chemistry involved the use of the silylating agent
N,O-bis(trimethylsilyl)acetamide (BSA), which has been
shown to induce amide bond formation between an unpro-
tected amino acid and an N-succinimide-activated ester.14
For the synthesis of xylariamide A we used the commercial
reagent 3-chloro-L-tyrosine (3), (E)-but-2-enedioic acid 2,5-
dioxo-pyrrolidin-1-yl ester methyl ester (4), and BSA in
DMF at 55 °C for 24 h (Scheme 1). Reaction workup, which
included C18 column chromatography and Sephadex LH-
20 gel permeation separation, afforded pure xylariamide
A (2, 47 mg, 48% yield). The NMR, MS, UV, and IR data
for the synthetic compound were essentially identical to
the natural product (1). However the optical rotation of
synthetic xylariamide A (2) ([R]23D +17° (CH3OH)) had the
Experimental Section
General Experimental Procedures. NMR spectra were
recorded at 30 °C on either a Varian 500 or 600 MHz Unity
INOVA spectrometer. The latter spectrometer was equipped
1
with a triple resonance cold probe. The H and 13C chemical
shifts were referenced to the solvent peak for DMSO-d6 at δ
2.49 and 39.51, respectively. LRESIMS were recorded on a
Fisons mass spectrometer. HRESIMS were recorded on a
Bruker Daltronics Apex III 4.7e Fourier transform mass
spectrometer. FTIR and UV spectra were recorded on a Perkin-
Elmer 1725X spectrophotometer and a GBC UV/vis 916
spectrophotometer, respectively. Optical rotations were re-
corded on a Jasco P-1020 polarimeter. Melting points were
determined on a Gallenkamp melting point apparatus and are
uncorrected. Sephadex LH-20 packed into an open glass
column (45 mm × 450 mm) was used for gel permeation
chromatography. A Waters AP-2 MPLC column (20 mm × 90
mm) packed with Alltech Davisil silica 30-40 µm 60 Å was
used for synthetic reaction purification. A Phenomenex Strata
Si-1 or C18-E SPE cartridge (1 g, 50 µm, 70 Å) was used for
synthetic reaction cleanup. A Waters 600 pump equipped with
a Waters 996 PDA detector and a Rheodyne injector were used
for HPLC. Thermo Hypersil C18 and phenyl BDS 5 µm 143 Å
preparative columns (21.2 mm × 150 mm) were used for HPLC
separations. All solvents used for chromatography, UV, and
MS were Lab-Scan HPLC grade, and the H2O used was
Millipore Milli-Q PF filtered. All synthetic reagents were
purchased from Sigma-Aldrich. All fungal culture media were
purchased from Difco.
opposite sign of the naturally occurring material (1) ([R]24
D
-22° (CH3OH)). Hence the synthetic material (+)-xylari-
amide A (2) was the enantiomer of the natural product,
and thus 3-chloro-D-tyrosine was determined to be a
constituent of (-)-xylariamide A (1).
Both synthetics 415 and 516,17 have been previously
synthesized; however both were only partially character-
ized by spectroscopic methods. 1D and 2D NMR experi-
ments were performed on both compounds in DMSO-d6,
and full NMR assignments were made. Initially, the
assignment of the 13C chemical shifts for C-2 and C-3 of 4
and 5 was not possible since both olefinic protons H-2 and
H-3 in 4 and 5 had identical or similar 1H chemical shifts
and they both showed strong HMBC correlations to the
carbonyl carbons C-1 and C-4. The full carbon NMR
assignments for 4 and 5 were made possible due to gHMBC

Notes
Journal of Natural Products, 2005, Vol. 68, No. 5 771
Fungal Material. The collection and identification of
7.07 (1H, d, J ) 16.0 Hz, H-3), 7.11 (1H, d, J ) 16.0 Hz, H-2);
13C NMR (125 MHz, DMSO-d6) δ 25.5 (2C, C-6, C-7), 52.6 (1-
OCH3), 126.9 (C-3), 137.9 (C-2), 163.9 (C-1), 160.6 (C-4), 169.8
(2C, C-5, C-8); (+)-LRESIMS m/z (rel int) 228 (20), 250 (100);
(+)-HRESIMS m/z 250.03208 (C9H9NO6Na [M + Na]+ requires
250.03221).
Xylaria sp. (FRR 5657) are detailed elsewhere.12
Fermentation, Extraction, and Isolation. The fungal
isolate was initially grown in three culture tubes each contain-
ing malt extract broth (10 mL) at 30 °C for 5 days. These
cultures were transferred to three conical flasks (500 mL) each
containing sterilized damp white rice (50 g of rice plus 100
mL of H2O), and the fermentation was allowed to proceed
under static conditions at 25 °C for 28 days. EtOAc extraction
of the cultures followed by removal of the solvent in vacuo
yielded a dark green gum (1.57 g). This extract was preab-
sorbed to C18 silica, then loaded onto a C18 flash column, and
a 20% stepwise gradient was performed from 100% H2O to
100% CH3OH. The 20% CH3OH/80% H2O elution was further
purified by C18 preparative HPLC using a linear gradient from
100% H2O to 50% CH3OH/50% H2O in 50 min at a flow rate
of 8 mL/min. Fraction 14 (11.6 mg, tR ) 38-41 min) was
subjected to phenyl preparative HPLC using a linear gradient
from 100% aqueous TFA (1.0%) to 100% CH3OH in 20 min at
a flow rate of 6 mL/min and yielded pure (-)-xylariamide A
(1, 0.9 mg, tR ) 17.2 min).
Synthesis of (+)-Xylariamide A (2). N,O-Bis(trimethyl-
silyl)acetamide (439 µL, 1.8 mmol) was added to (E)-but-2-
enedioic acid 2,5-dioxo-pyrrolidin-1-yl ester methyl ester (4,
68 mg, 0.3 mmol) and 3-chloro-L-tyrosine (3, 195 mg, 0.9 mmol)
in dry DMF (3 mL), and the reaction was heated at 55 °C for
24 h. Upon cooling, the DMF was removed in vacuo and the
residue was preabsorbed to C18. The C18 material was added
to a C18 SPE cartridge, and 20% stepwise elutions were run
from 100% aqueous TFA (0.1%) to 100% CH3OH (10 mL
washes). Fractions 3 and 4 were combined, then subjected to
Sephadex LH-20 chromatography using 100% CH3OH as the
eluent at a flow rate of 4.5 mL/min. All resulting fractions were
analyzed by TLC and identical fractions combined to yield pure
(+)-xylariamide A (2, 47 mg, 48% yield) as a stable yellow gum:
[R]23 +17° (c 0.313, CH3OH); UV (CH3OH) λmax (log ꢀ) 207
D
(-)-Xylariamide A (1): stable yellow gum; [R]24 -22° (c
D
(4.18), 218 (sh) (4.08), 278 (3.51) nm; IR νmax (NaCl) 3400-
0.060, CH3OH); UV (CH3OH) λmax (log ꢀ) 202 (4.05), 217 (sh)
(3.74), 278 (3.12) nm; IR νmax (NaCl) 3600-3100, 1716, 1667,
3100, 1716, 1667, 1541, 1512, 1437, 1293, 1227, 1197, 1167,
1
1056, 1020, 977, 818 cm-1; H NMR (500 MHz, DMSO-d6) δ
1
1544, 1511, 1438, 1293, 1197, 1024, 975, 826 cm-1; H NMR
2.77 (1H, dd, J ) 14.0, 9.5 Hz, H-7a), 3.00 (1H, dd, J ) 14.0,
4.5 Hz, H-7b), 3.71 (3H, s, 13-OCH3), 4.44 (1H, ddd, J ) 9.5,
8.5, 4.5 Hz, H-8), 6.54 (1H, d, J ) 16.0 Hz, H-12), 6.85 (1H, d,
J ) 8.5 Hz, H-5), 6.97 (1H, d, J ) 8.5 Hz, H-6), 7.06 (1H, d,
J ) 16.0 Hz, H-11), 7.16 (1H, s, H-2), 8.77 (1H, d, J ) 8.5 Hz,
8-NH), 10.08 (1H, brs, 4-OH); 13C NMR (125 MHz, DMSO-d6)
δ 35.6 (C-7), 52.0 (13-OCH3), 54.1 (C-8), 116.4 (C-5), 119.1 (C-
3), 128.5 (C-12), 128.6 (C-6), 129.1 (C-1), 130.2 (C-2), 137.1 (C-
11), 151.6 (C-4), 162.6 (C-10), 165.4 (C-13), 172.4 (C-9); (-)-
LRESIMS m/z (rel int) 168 (20), 167 (7), 282 (10), 284 (3), 326
(100), 328 (33); (-)-HRESIMS m/z 326.04419 (C14H13NO635Cl
[M - H]- requires 326.04369).
(500 MHz, DMSO-d6) δ 2.77 (1H, dd, J ) 14.5, 10.0 Hz, H-7a),
3.00 (1H, dd, J ) 14.5, 5.0 Hz, H-7b), 3.71 (3H, s, 13-OCH3),
4.46 (1H, ddd, J ) 10.0, 8.0, 5.0 Hz, H-8), 6.54 (1H, d, J )
15.5 Hz, H-12), 6.84 (1H, d, J ) 8.5 Hz, H-5), 6.97 (1H, dd,
J ) 8.5, 1.5 Hz, H-6), 7.05 (1H, d, J ) 15.5 Hz, H-11), 7.17
(1H, d, J ) 1.5 Hz, H-2), 8.80 (1H, d, J ) 8.0 Hz, 8-NH), 9.95
(1H, brs, 4-OH); 13C NMR (125 MHz, DMSO-d6) δ 35.5 (C-7),
52.0 (13-OCH3), 53.8 (C-8), 116.4 (C-5), 119.2 (C-3), 128.6 (C-
12), 128.6 (C-6), 128.9 (C-1), 130.2 (C-2), 136.9 (C-11), 151.6
(C-4), 162.7 (C-10), 165.4 (C-13), 172.3 (C-9); (-)-LRESIMS
m/z (rel int) 168 (15), 167 (5), 282 (10), 284 (3), 326 (100), 328
(33); (-)-HRESIMS m/z 326.04257 (C14H13NO635Cl [M - H]-
requires 326.04369).
Brine Shrimp Lethality Assay. Brine shrimp eggs were
obtained from Bio-Marine Aquafauna and were hatched in
artificial seawater prepared from Instant Ocean (Aquarium
Systems). The hatching, harvesting, and dispensing of nauplii
were performed in a manner similar to that described by Solis
et al.19 Compounds 1 and 2 were added to live nauplii (10-20
per vial, VT ) 500 µL) in glass vials (2 mL), and following 24
h of drug treatment the brine shrimp lethality was measured
by counting the number of dead (nonmotile) nauplii per vial.
Compounds 1 and 2 were tested in triplicate at 20 and 200
µg/mL and were solubilized in 100% DMSO with a final DMSO
concentration of 2% in each vial. The brine shrimp responded
typically when treated with the laboratory standards, lisso-
clinotoxins E and F.20 For example, the LD50 of lissoclinotoxin
F toward the brine shrimp was ∼5 µg/mL.
Synthesis of (E)-But-2-enedioic Acid Monomethyl Es-
ter (5). The commercial reagent (E)-but-2-enedioic acid di-
methyl ester (6, 1.44 g, 10 mmol) was dissolved in acetone (70
mL) at room temperature, and 1 N LiOH (10 mL, 10 mmol)
was slowly added over 15 min to the stirred solution. The
reaction was stirred for 1 h, diluted with 2 N HCl (200 mL),
saturated with NaCl, and then extracted with EtOAc (3 × 200
mL). The EtOAc layer was slowly evaporated, and the result-
ing precipitate was filtered and dried to yield pure (E)-but-2-
enedioic acid monomethyl ester (5, 1.13 mg, 87% yield) as a
white amorphous solid: mp 142-143 °C (lit. mp 141-141.5
°C);16,17 UV (CH3OH) λmax (log ꢀ) 209 (4.15) nm; IR νmax (NaCl)
1803, 1775, 1751, 1656, 1441, 1425, 1362, 1314, 1286, 1204,
1
1101, 1049, 977, 962, 886, 809, 759, 736, 651 cm-1; H NMR
(500 MHz, DMSO-d6) δ 3.72 (3H s, 1-OCH3), 6.69 (2H, s, H-2,
H-3), 13.18 (1H, brs, 4-OH); 13C NMR (125 MHz, DMSO-d6) δ
52.3 (1-OCH3), 132.3 (C-2), 134.7 (C-3), 165.1 (C-1), 165.7 (C-
4); (-)-LRESIMS m/z (rel int) 129 (100); (-)-HRESIMS m/z
129.01930 (C5H5O4 [M - H]- requires 129.01933).
Acknowledgment. The author thanks the Eskitis Insti-
tute and Griffith University (NRG) for research funding, and
Drs. J. I. Pitt, A. Hocking, and N. Tran-Dinh from Food Science
Australia for fungal identification. Dr. J. Mitchell from Griffith
University is acknowledged for obtaining the HRESIMS data.
Synthesis of (E)-But-2-enedioic Acid 2,5-Dioxo-pyrro-
lidin-1-yl Ester Methyl Ester (4). (E)-But-2-enedioic acid
monomethyl ester (5, 260 mg, 2 mmol), EDCI (768 mg, 4
mmol), and N-hydroxysuccinimide (690 mg, 6 mmol) were
dissolved in dry CH3CN (5 mL), and the reaction was stirred
at room temperature for 24 h. The reaction mixture was
preabsorbed to silica, then loaded onto a glass column and
flushed with 100% EtOAc (50 mL). The EtOAc wash was
evaporated to dryness, redissolved in 100% DCM, and injected
onto a MPLC silica-packed column using isocratic conditions
of 40% EtOAc/60% hexanes at a flow rate of 6 mL/min for 30
min. This yielded pure (E)-but-2-enedioic acid 2,5-dioxo-
Supporting Information Available: 1H and 13C NMR spectra
and LRESIMS data for compounds 1, 2, 4, and 5. This material is
available free of charge via the Internet at http://pubs.acs.org.
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772 Journal of Natural Products, 2005, Vol. 68, No. 5
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