Antimicrobial metabolites produced by an intertidal Acremonium furcatum

Article abstract of DOI:10.1016/j.phytochem.2006.07.028

In a screening for antimicrobial metabolites, amides of d-allo- and l-isoleucine derivatives were isolated from the culture of a marine strain of Acremonium furcatum. Structural elucidation of these compounds was performed by analysis of spectroscopic data and confirmed by synthesis. All of the compounds, natural and synthetic intermediates, were bioassayed against bacteria and phytopathogenic fungi, with many showing remarkable antifungal activities.

Full text of DOI:10.1016/j.phytochem.2006.07.028

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 2403–2410
www.elsevier.com/locate/phytochem
Antimicrobial metabolites produced by an intertidal
Acremonium furcatum
a
a
a
b
´
´
Gabriela L. Gallardo , Matıas Butler , Mariana L. Gallo , M. Alejandra Rodrıguez ,
c
a,
*
Marcos N. Eberlin , Gabriela M. Cabrera
a
Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Pab. II, 1428 Buenos Aires, Argentina
b
DBBE, FCEN, UBA, Ciudad Universitaria, Pab. II, 1428 Buenos Aires, Argentina
Thomson Mass Spectrometry Laboratory, Instituto de Quı´mica, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil
c
Received 13 January 2006; received in revised form 27 June 2006
Available online 7 September 2006
Abstract
In a screening for antimicrobial metabolites, amides of D-allo- and L-isoleucine derivatives were isolated from the culture of a marine
strain of Acremonium furcatum. Structural elucidation of these compounds was performed by analysis of spectroscopic data and con-
firmed by synthesis. All of the compounds, natural and synthetic intermediates, were bioassayed against bacteria and phytopathogenic
fungi, with many showing remarkable antifungal activities.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Acremonium furcatum; Marine fungi; Isoleucinol; Isoleucinaldehyde; Isoleucine derivatives; Antifungal activity; Fusarium virguliforme; Coll-
etotrichum truncatum
1. Introduction
deals with the fermentation, isolation, structure elucida-
tion, synthesis and biological activity of these compounds,
which are amides of the corresponding amino alcohols and
amino aldehydes of D-allo- and L-isoleucine.
Since marine microorganisms grow in unique and
extreme habitats, they may have the capability to produce
unique and unusual metabolites. In particular, it is known
that marine fungi, native or adapted to the environment,
are an important source of bioactive natural products
(Schiehser et al., 1986; Fenical and Jensen, 1993; Kobay-
ashi and Ishibashi, 1993; Davidson, 1995; Bringmann
et al., 2005). In the course of screening for new bioactive
metabolites with potential use in agriculture from fungal
cultures of diverse origin (Cabrera et al., 2002; Gallo
et al., 2004; Levy et al., 2003), we investigated a strain
Acremonium furcatum isolated from an intertidal sediment
sample. The extract of the culture medium of this fungus
produced new metabolites with antimicrobial activity
against bacteria and phytopathogenic fungi. This report
2. Results and discussion
The organic extract of the culture media of A. furcatum,
isolated from an intertidal sediment sample (collected on
the coast of Buenos Aires, Argentina), showed moderate
antimicrobial activity against Bacillus subtilis, Staphylococ-
cus aureus, Escherichia coli and Botrytis cynerea, and a very
simple pattern by thin layer chromatography (TLC). Prep.
TLC was then employed and two fractions in a 10:1 ratio
were obtained. The major and more polar fraction showed,
1
by H NMR spectroscopy, three olefinic protons at d 6.88
(1H, d, 11.0 Hz), 6.33 (1H, ddq, 15.0, 11.0 and 1.5 Hz) and
6.03 (1H, dq, 15.0 and 6.9 Hz), one broad signal at
5.90 ppm, five complex multiplets at 4.00, 3.70, 1.70,
*
Corresponding author. Tel./fax: +54 11 4576 3376.
E-mail address: gabyc@qo.fcen.uba.ar (G.M. Cabrera).
0031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.07.028

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G.L. Gallardo et al. / Phytochemistry 67 (2006) 2403–2410
1.46–1.50 and 1.23 ppm, two methyl protons attached to
sp2 carbons at d 1.86 (d, 6.9 Hz) and 1.95 (s), and two set
of methyl groups in a 3:1 ratio, the major group at d 0.93
(t, 7.4 Hz) and 0.93 (d, 7.0 Hz) and the minor at 0.91 (t,
7.0 Hz) and 0.95 (d, 6.8 Hz). These data indicated that
the fraction was actually a mixture of two compounds 1
and 2, in an approximately 3:1 ratio, which were very sim-
ilar in nature. The high field signals resembled those of
amino acids and the similarity between both compounds
suggested an epimeric mixture.
Exhaustive efforts to separate this mixture using HPLC,
employing different stationary and mobile phases, or
employing enzymes to selectively acylate one epimer (after
knowing the structures), were only partially successful – the
major compound 1, obtained via HPLC separation with a
chiral stationary phase, was only of sufficient purity for full
characterization. Thus, a provisional structural elucidation
of compound 2 was performed on the natural mixture (Che
et al., 2002).
Compound 1 has the molecular formula C13H23NO2, as
determined from its high-resolution electron ionization mass
spectrometry (EIMS). A COSY H,H experiment and irradi-
ation of the olefinic protons allowed unambiguous determi-
nation of the presence of a CH3CH@CHACH@C(CH3)
moiety and, taking into account the coupling constants, an
E configuration for the terminal double bond was deter-
mined. The COSY spectrum also showed that the methine
proton at d 4.00 (CH) correlated with protons at d 3.70
(CH2) and 1.72 (CH).
spectroscopic literature data for the corresponding methyl
´
ester, previously synthesized (Ceroni and Sequin, 1982).
A comparison of the remaining high-field proton and
carbon signals with the literature data for amino acids
(MacDonald et al., 1976) showed that compound 1 con-
tained a substructure similar to the amino acid isoleucine,
but in a reduced way, isoleucinol, as 2D experiments
revealed. The connectivities observed in the HMBC exper-
iment are shown in Fig. 2. The structure of compound 1
was thus assigned as the 2-methyl-hexa-2,4-dienoic acid,
isoleucinol amide.
Similar spectroscopic analysis was performed on the
mixture, revealing the presence of a minor component with
the same structural features as 1. The 2D NMR spectro-
scopic experiments showed that all the correlations in the
high field region were merely duplicated and partially over-
lapped. Furthermore, the available 13C NMR spectro-
scopic data for amino acids (MacDonald et al., 1976)
suggested that 1 and 2 were a 3:1 mixture of C-2 epimers,
i.e. D-allo-IleOH:L-IleOH or L-allo-IleOH:D-IleOH, indicat-
ing that the major epimer was either D-allo or L-allo-IleOH,
whereas the minor component was L- or D-IleOH.
The mixture of compounds1 and 2 was oxidized and
hydrolyzed to determine the absolute configuration of each
compound, with the resulting amino acids analyzed by
HPLC using a chiral column, Chirex (D) Penicillamine,
yielding peaks with retention times of 29.9 and 39.9 min,
identical to authentic samples of D-allo-Ile and L-Ile, respec-
tively. This result thus established the absolute configura-
tion as being 20R, 30S for the major epimer 1, and 20S, 30S
for the minor compound 2. With this rationale, the struc-
tures of 1 and 2 were established as shown in Fig. 1.
The synthesis of compounds 1 and 2 was performed to
confirm the structures, to determine the physical constants
of compound 2, and to fundamentally evaluate the bio-
logical properties of each individual compound. For this
The 13C NMR spectrum exhibited four olefinic carbons
(d 136.3 d, 134.2 d, 127.3 s and 126.9 d) and two methyl
groups at 18.6 and 12.7 ppm, corresponding to the
above-mentioned olefinic moiety, plus another seven sig-
nals, which were all accompanied by a duplicate resonance
of lower intensity in the 13C NMR spectrum of the mixture.
One of these carbons was a carbonyl at 170.3 ppm, while
the other carbons were a CH2 attached to an oxygen at d
64.0, a CH linked to a heteroatom at 55.3 ppm and ali-
phatic carbons at 35.5 (d), 26.3 (t), 14.8 and 11.3 (q). The
multiplicities were determined by a distortionless enhance-
ment by polarization transfer (DEPT) experiment.
OH
O
The methyl group at 1.95 ppm correlated with the car-
bonyl in a heteronuclear multiple bond correlation (HMBC)
experiment, indicating that an acid derivative CH3CH@
CHACH@C(CH3)COX was present and attached to the
other part of the molecule. The E configuration for the sec-
ond double bond was determined by comparison with the
NH
Fig. 2. Observed HMBC correlations.
OH
1´
O
O
CHO
5
4´
3
2´
3´
6´
1
NH
5´
6
NH
2
4
7
1 2´R
2 2´S
3 2´R
4 2´S
Fig. 1. Isolated compounds from Acremonium furcatum.

G.L. Gallardo et al. / Phytochemistry 67 (2006) 2403–2410
2405
OH
purpose, the general methodology of Hamada et al.
CHO
COOMe
(1987) was employed. Although some modifications were
made to improve yields, in general, they were lower due
to simple decomposition of the unsaturated acid subunit.
This subunit was synthesized instead according to the
a
b
BOCNH
10
BOCNH
BOCNH
9
a. LiBH4/EtOH b. PCC/CH2Cl2
´
procedure of Ceroni and Sequin (1982) as shown in
Fig. 4. Synthetic route to compound 10.
Fig. 3. The spectroscopic data of the synthetic compounds
1 and 2 were in full accordance with those of the natural
metabolites.
100
80
60
40
20
0
The minor and less polar fraction isolated from the
1
extract had similar H and 13C NMR spectra that of com-
pounds 1 and 2. It differed, however, by the absence of the
CH2OH moiety and the presence of an aldehydic function-
1
ality at d 200.2 (13C NMR) and 9.73 and 9.67 ppm (s, H
NMR), respectively, suggesting structures 3 and 4 as shown
in Fig. 1. An epimeric mixture was also evident, as the sig-
nals of the aminoaldehyde unit were duplicated in both
spectra. Due to the small amount of this mixture, and its
instability, it was not possible to perform any further anal-
ysis. The presence of the epimers, D-allo- and L-Ile-isoleu-
cinaldehyde, was thus assumed on the basis of the
absolute stereochemistry of 1 and 2. The synthesis of
N-Boc-L-isoleucinaldehyde 10 (Fig. 4) was made to confirm
the assignments and identify unambiguously both epimers.
0
5
10
15
20
25
Time (days)
1
The H and 13C NMR spectroscopic data for 10 were in
pH x 10
full agreement with those of the minor epimer 4, confirm-
ing its identity.
Freeze-dried mycelium (mg/5 ml)
Compounds 1 + 2 (mg/l)
A time-course of the production of compounds 1 + 2
was carried out in an attempt to explain the reason why
both epimers were present in the extract (Fig. 5). Two inter-
esting findings were made. First, the pH of the medium
became basic after day 8. Second, the presence of the minor
metabolites 3 and 4 were observed on all days, via TLC of
the extract, in approximately the same relative proportions
to that of the epimers 1 and 2. Both results thus suggested
that the aldehydic precursors of the alcohols epimerize due
to a keto–enol equilibrium in the basic media, thereby pro-
ducing an epimeric mixture of alcohols.
Fig. 5. Time-course production of 1 and 2.
soy bean) Colletotrichum truncatum (responsible for
anthracnose disease), Macrophomina phaseolina (causal
agent of charcoal rot in beans), B. cynerea and Aspergillus
fumigatus. The results are shown in Table 2. Interestingly,
the L-derivatives 2 and 8 showed moderate antibiotic activ-
ity and low, if any, antifungal activity, whilst the D-allo
derivatives 1 and 7 showed antifungal activity. Nonethe-
less, the acid 6 and the ester 5 displayed the greatest anti-
The synthetic compounds 1 and 2, and the synthetic
intermediates 5–8 and 10, were bioassayed against the bac-
teria B. subtilis, S. aureus, E. coli and the fungi Fusarium
virguliforme (causal agent of Sudden Death Syndrome in
fungal activity, in the same order as benomyl, a
commercial antifungal product. The natural (2Z, 4E) iso-
mer of 6 was previously reported as an antifungal metabo-
lite against other fungal strains (Proksa et al., 1992).
O
OH
O
O
O
a
b
Br
+
H
OCH2CH3
OCH2CH3
OCH2CH3
6
c
1
O
CH2OH
O
CO2CH3
O
d
e
1´
NH
NH
2
OH
5
6´
7´
1´ 2R
2´ 2S
7
8 2R
9 2S
a. Zn/Bz b. P2O5/silicagel c. NaOH/t-BuOH d. Cl H3NCHRCOOMe , Py, DCC/THF e. LiBH4/EtOH
Fig. 3. Synthetic route to compounds 1 and 2.

2406
G.L. Gallardo et al. / Phytochemistry 67 (2006) 2403–2410
Table 1
phersen et al., 1999) although this question deserves still an
explanation.
NMR spectroscopic data of compounds 1 and 2 (CDCl3), d in ppm, J in
Hz
Acremonium is a wide-spread fungal genus, noted for
their secondary metabolite content, with around 90 com-
pounds having been reported from fungi of this genus
(Abdel-Lateff et al., 2002), including alkaloids (Munday-
Finch et al., 1995), terpenoids (Kawashima et al., 1994),
aromatic compounds (Toki et al., 1994) and peptides
(Sharman et al., 1996). Particularly, several marine Acre-
monium strains have been isolated, including strains of A.
furcatum (Koh et al., 2002), and antioxidant hydroqui-
nones (Abdel-Lateff et al., 2002), anti-inflammatory oxe-
pines and weak antifungal quinolines (Belofsky et al.,
2000) were isolated and identified from marine strains of
this genus.
Compound 1
Compound 2
13C
1H
13C
1H
1
2
3
4
170.3
127.3
134.2
126.9
170.1
126.9
134.2
126.9
6.88 d (11.0)
6.33 ddq
6.88 d (11.0)
6.33 ddq
(15.0, 11.0, 1.5)
6.03 dq (15.0, 6.9)
1.86 d (6.9)
1.95 s
3.70 dd (11.2, 4.1)
3.67 dd (11.2, 6.5)
4.00 m
(15.0, 11.0, 1.5)
6.03 dq (15.0, 6.9)
1.86 d (6.9)
1.95 s
5
6
7
10
136.3
18.6
12.7
64.0
136.3
18.6
12.7
63.4
3.74 ma
20
55.3
35.5
26.3
11.3
14.8
55.9
35.7
25.5
11.3
15.5
3.88 m
1.70 m
1.23, 1.51 m
0.91 t (7.0)
0.95 d (6.8)
5.92b
30
1.72 m
40
1.23, 1.46 m
0.93 t (7.4)
0.93 d (7.0)
5.90 d (7.7)
50
2.1. Concluding remarks
60
NH
In summary, we have isolated and identified two new
antimicrobial amino alcohol derivatives 1 and 2, which
are epimers biosynthesized from the corresponding alde-
hydes, that were also isolated. A rapid epimerization of
the aldehydes could explain these data, as a basic pH was
produced in the medium during fermentation. The struc-
ture elucidations were confirmed by total synthesis of com-
pounds 1 and 2, and remarkably, the two synthetic
intermediates 5 and 6, exhibited higher antifungal activity
against phytopathogenic fungi, comparable to that of the
commercial agent benomyl.
Assignments based on COSY H,H, HSQC and HMBC experiments.
Multiplicities determined by DEPT.
a
Overlapped.
Partially overlapped.
b
Several isoleucine derivatives were previously isolated
from different microbial strains as C-amides (Kern et al.,
1985), esters (Iwamoto et al., 1990) or unknowns (Kawa-
kami et al., 1978). In particular, isoleucinol was reported
as a C-terminal amino acid of peptaibols (Jaworski and
Bruckner, 2001).
The acid subunit was previously found as an ester of an
antifungal compound isolated from Graphium putredinis
(Kennedy et al., 1998) and the above-mentioned antifungal
isomer (2Z, 4E) was identified as a free acid from culture
extracts of Penicillium vermiculatum (Proksa et al., 1992).
It is noteworthy that, although the strain grew well in the
same medium without artificial sea water and also in malt
extract agar, the compounds were not produced, even at
trace levels, under those conditions. It is known that
metabolite profiles expressed by fungi are sometimes
dependent on media salinity (Rabaek et al., 1998; Christo-
3. Experimental
3.1. General
FTIR spectra were recorded on a Nicolet Magna-IR
550. The UV spectra were recorded on a Hewlett–Packard
8451 A diode array spectrophotometer, whereas optical
rotations were acquired on a Perkin–Elmer polarimeter
343. NMR spectra were recorded on a Bruker AM-500
1
instrument at 500.13 MHz for H and at 125.13 MHz for
Table 2
Antimicrobial activities of natural and synthetic compounds
1
2
5
6
7
8
10
A
B
Escherichia colia
–
–
7
15
15
15
10
nd
7
9
–
10c
10c
–
–
15
15
–
9
9
25 (1 lg)
29 (1 lg)
15 (1 lg)
12 (1 lg)
20 (1lg)
–
–
–
15
10
–
8
nd
7
–
–
–
–
14
–
11
nd
9
10
8
12
10
12
nd
nd
nd
nd
nd
20
20
28
25
22
Bacillus subtilisa
10
–
12c
–
–
–
Staphylococcus aureusa
Fusarium virguliformeb
Aspergillus fumigatusb
Botrytis cynereab
Colletotrichum truncatumb
Macrophomina phaseolinab
15 (5 lg)
24 (1 lg)
12 (5 lg)
10 (10 lg)
10 (25lg)
5
nd
nd
Diameter of inhibition zone in mm (MIC lg/pt).
nd, not determined.
a
50 lg/6 mm disk was used.
b
50 lg/spot was used except the concentration shown in parentheses and B: benomyl (25 lg/spot). A: a mixture of natural products 1 and 2.
Diffuse halos.
c

G.L. Gallardo et al. / Phytochemistry 67 (2006) 2403–2410
2407
13C NMR. EIMS was carried out on a mass spectrometer
Trio-2 VG Masslab (Manchester, UK), whereas HR-MS
employed a Fisons VG AutoSpec. Some of the HR-MS
were run at the Washington University Resource (St.
Louis, Missourri) for Biomedical and Bio-organic Mass
Spectrometry.
determined by HPLC (Column: YMC-Pack ODS-A,
250 · 4.6 mm, 5 lm; MeOH–H2O 6:4, 0.5 ml/min, UV
230 nm).
3.6. Physico-chemical properties of compounds
3.6.1. Compound 1
3.2. Fungal strain
(2E,4E)-2-methyl-hexa-2,4-dienoic acid (20R,30S)-isoleu-
cinol amide or ((2E,4E)-2-methyl-hexa-2,4-dienoic acid
[(10R,20S)-1-hydroxymethyl-2-methyl-butyl]-amide). Oil.
[a]D = ꢀ50 (CHCl3, c0.1). For 1H NMR and 13C NMR spec-
tra, see Table 1. HREIMS m/z [M]+Æ, found 225.17300, calc.
for C13H23NO2 225.17288 (Dm = 0.4 ppm), [M + 1]+Æ,
found 226.17656, calc. for 13C12C12H23NO2 226.17623 (D
m = 0.4 ppm). EIMS 70 eV, m/z (rel. int.): 225 [M]+Æ(8),
210 [M ꢀ CH3]+ (3), 207 [M ꢀ H2O]+Æ (3), 194
[M ꢀ CH2OH]+Æ (24), 126 (22), 109 [RCO]+ (100), 81 (69).
The fungus A. furcatum (F. & R. Moreau) ex W. Gams,
was isolated from an intertidal marine sediment sample col-
lected at Miramar, Province of Buenos Aires, Argentina
and classified by Dr. J. E. Wright (PRHIDEB-CONICET,
´
Depto de Biodiversidad y Biologıa Experimental DBBE,
FCEN, UBA) and Dr. M. A. Rodriguez (DBBE, FCEN-
UBA). The strain was deposited in the BAFC Culture Col-
lection (FCEN-UBA, CONICET) under the Accession
Number BAFC 51375.
3.6.2. Compound 2
3.3. Fermentation
(2E,4E)-2-methyl-hexa-2,4-dienoic acid (20S,30S)-isoleu-
cinol amide or ((2E,4E)-2-methyl-hexa-2,4-dienoic acid
[(10R,20S)-1-hydroxymethyl-2-methyl-butyl]-amide). For
1H NMR and 13C NMR spectra (in the natural mixture),
see Table 1.
Fermentation was carried out using a medium which
consisted of peptone 1%, yeast extract 0.5%, dextrose 1%
and artificial sea water (Instant Ocean) 100%. Erlenmeyer
flasks (250 ml) containing 75 ml of medium were inocu-
lated with agar slants of the strain. Fermentation was car-
ried out at 25 ꢁC for 20 days under static conditions.
3.6.3. Compounds 3 and 4
(20R, 30S) and (20S,30S) [(2E,4E)-2-Methyl-hexa-2,4-die-
noic acid isoleucinaldehyde] or ((10R,20S) and (10S,20S)
3.4. Extraction and isolation
(2E,4E)-2-methyl-hexa-2,4-dienoic
acid
(1-formyl-2-
methyl-butyl)-amide). 1H NMR (500 MHz, CDCl3): d
9.73 (s, H-10m), 9.67 (s, H-10M), 6.89 (m, H-3), 6.34 (m, H-
4), 6.06 (m, H-5), 5.85 (NH), 3.73 (m, H-20), 1.96 (s, H-7),
1.87 (d, J = 6.4 Hz, H-6), 1.30–40 (m, H-40), 0.94 (t,
J = 6.9 Hz, H-50M), 0.94 (d, J = 6.6 Hz, H-60M), 0.99 (d,
J = 7.0 Hz, H-6m0 ), 0.97 (overlapped, H-50m). 13C NMR
(125 MHz, CDCl3): d 200.2 (d, C-10), 169.5 (s, C-1M),
169.2 (s, C-1m), 136.5 (d, C-5), 134.4 (d, C-3), 127.0 (C-
2,4), 62.0 (d, C-20M), 63.1 (d, C-2m0 ), 36.5 (d, C-30m), 35.5 (d,
C-30M), 26.4 (t, C-4M0 ), 25.7 (t, C-40m), 18.8 (q, C-6), 15.6 (q,
C-60m), 14.8 (q, C-60M), 12.8 (q, C-7), 11.9 (q, C-50). M: major
epimer 3 (20R,30S), m: minor epimer 4 (20S,30S).
The fermentation broth was filtered and the filtrate was
partitioned with EtOAc. The crude organic extract was sub-
jected to prep. TLC on silica gel (0.25 mm), eluting with
EtOAc:CH2Cl2 1:1, yielding one band containing com-
pounds 1 and 2 (29 mg/l, RF 0.5) and another with com-
pounds 3 and 4 (1 mg/l, RF 0.7). Both bands were
detected by UV at 254 nm, and EtOAc was employed to
recover the compounds from the adsorbent. Compounds
1 and 2 were separated by HPLC (Chirex (D) Penicillamine
column-Phenomenex, 250 · 4.60 mm, CuSO4 1 mM:MeOH
6:4, 0.7 ml/min, UV 215 nm). A re-purification of the major
peak by peak shaving was done employing the same condi-
tions as above to yield pure 1 (1 mg).
3.7. Absolute configuration determination
3.5. Time-course of production of compounds 1 and 2
3.7.1. Oxidation of compounds 1 and 2 to isoleucine
A solution of 5 mg of a sample of compounds 1 and 2 in
acetone was oxidized with Jones reagent at ambient tem-
perature (Harding et al., 1975). The product mixture was
then subjected to TLC (CH2Cl2–MeOH 85:15) yielding
the corresponding dicarbonylated intermediates (3-methyl-
A loopful of the cells of a slant culture of the strain
was inoculated in 75 ml of the above liquid medium in
a 250 ml Erlenmeyer flask and incubated on a rotary sha-
ker at room temperature for one day to give a seed cul-
ture. An aliquot of this culture (1 ml) was transferred
quantitatively onto 50 ml of medium in 40 · 125 ml Erlen-
meyer flasks. Two of these cultures were filtered every day
with the mycelia freeze-dried and the media extracted with
EtOAc (20 ml); the aqueous layers were re-extracted with
EtOAc (2 · 20 ml). The organic layers were combined and
evaporated in vacuo with the amount of compounds 1 + 2
2-(2-oxo-propionylamino)-pentanoic
acid).
1HNMR
(500 MHz, CD3OD-CDCl3 1:1): d 4.51 (d, J = 4.0 Hz, H-
aM), 4.40 (d, J = 4.8 Hz, H-am), 2.47 (CH3CO), 0.95 (t,
J = 6.9 Hz, H-5M), 0.92 (d, J = 6.6 Hz, H-6M); M: major
epimer, m: minor epimer. The intermediates were hydro-
lyzed with 6 N HCl at 110 ꢁC for 16 h, with the product
neutralized and directly used for HPLC analysis.

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G.L. Gallardo et al. / Phytochemistry 67 (2006) 2403–2410
The absolute configuration determination of the amino-
product was concentrated in vacuo, and was directly
employed for the next step. It was dissolved in THF
(10 ml) together with compound 6 (173 mg) and 1 equiv.
of pyridine. The mixture was then cooled in an ice bath
and dicyclohexylcarbodiimide (DCC) (300 mg) was added.
After 30 min, the reaction was allowed to proceed for 4 h at
room temperature. The dicyclohexylurea was removed by
filtration (Klausner and Bodanski, 1972). The reaction
mixture was evaporated to dryness and the crude product
(150 mg) was purified by dry column flash chromatogra-
phy, eluting fractions with mixtures of cyclohexane–
CH2Cl2 1:1 to CH2Cl2–EtOAc 1:1 of increasing polarity.
Pure compound 7 was eluted with CH2Cl2. Compound 7:
Oil. [a]D = ꢀ50 (CH2Cl2, c0.49). UV (CH2Cl2)kmax (nm)
(loge): 240 (4.7). FTIR (KBr) mmax (cmꢀ1): 3408 (NH),
2968 (CH), 2920 (CH), 2860 (CH), 1763 (CO), 1669,
acids, derived from 1 and 2, was done by HPLC (Chirex
(D) Penicillamine column-Phenomenex, 250 · 4.60 mm,
CuSO4 2 mM: MeOH 85:15, 0.7 ml/min, UV 215 nm) by
comparison with authentic standards of amino acids. RT
values: L-allo-Ile 25.6 min, L-Ile 29.9 min, D-allo-Ile
39.9 min, D-Ile 49.5 min. Sample from 1 and 2: 29.9 and
39.9 min. Coinjections confirmed the identities.
3.8. Synthesis of compounds 1 and 2
3.8.1. Synthesis of (2E,4E)-2-methyl-hexa-2,4-dienoic acid
ethyl ester (5)
This compound was prepared according to the previous
reported procedure for the corresponding methyl ester
´
(Ceroni and Sequin, 1982). Instead of the use of Sicapent
1
drying agent, P2O5 on Silicagel Kieselgel 60 G-Merck
(1:2) was employed. Compound 5: Oil. UV (CH2Cl2)kmax
nm (loge): 236 (3.4). FTIR (KBr) mmax cmꢀ1: 2981 (CH),
2931 (CH), 1740 (CO), 1455, 1376, 1241, 1177, 1099, 950.
1H NMR (CDCl3): d 7.15 (br d, J = 11.4 Hz, H-3), 6.36
(ddq, J = 15.0, 11.4 and 1.6 Hz, H-4), 6.08 (dq, J = 15.0
and 6.8 Hz, H-5), 4.20 (q, J = 7.0 Hz, CH2CH3), 1.92 (br
s, H-7), 1.86 (br d, J = 6.8 Hz, H-6), 1.29 (t, J = 7.0 Hz,
CH2CH3). 13C NMR (CDCl3): d 168.4 (s, C-1), 138.2 (d,
C-3), 137.3 (d, C-5), 127.3 (d, C-4), 124.8 (s, C-2), 60.2 (t,
CH2CH3), 18.6 (q, C-6), 14.1 (q, CH2CH3), 12.3 (q, C-7).
EIMS 70 eV m/z (rel. int.): 155 [M + 1]+ (30), 154 [M]+Æ
(21), 127 (21), 126 (8), 99 (45), 81 (50), 55 (100).
1520, 1467. H NMR (CDCl3): d 6.88 (br d, J = 11.1 Hz,
H-30), 6.33 (ddq, J = 14.9, 11.1 and 1.6 Hz, H-40), 6.18
(br d, J = 8.7 Hz, NH), 6.04 (dq, J = 14.9 and 6.9 Hz, H-
50), 4.80 (dd, J = 8.7 and 4.1 Hz, H-2), 3.75 (s, CO2CH3),
1.96 (br s, H-70), 1.97 (overlapped m, H-3), 1.86 (br d,
J = 6.9 Hz, H-60), 1.45 and 1.19 (m, H-4), 0.96 (t,
J = 7.3 Hz, H-5), 0.90 (d, J = 6.8 Hz, H-6). 13C NMR
(CDCl3): d 173.1 (s, C-1), 169.1 (s, C-10), 136.4 (d, C-50),
134.3 (d, C-30), 127.3 (s, C-20), 127.0 (s, C-40), 55.5 (d, C-
2), 52.1 (s, CO2CH3), 38.0 (d, C-3), 26.3 (t, C-4), 18.7 (q,
C-60), 14.7 (q, C-6), 12.8 (q, C-70), 11.7 (q, C-5). HREIMS
m/z: (M)+Æ, found 253.16753, calc. for C14H23NO3
253.16779 (Dm = 1.0 ppm). EIMS 70 eV, m/z (rel. int.):
253 [M]+Æ(24), 238 (15), 144 (17), 130 (37), 128 (7), 109
[RCO]+ (100), 86 (50), 81 (24).
3.8.2. Hydrolysis of 5 to (2E,4E)-2-methyl-hexa-2,4-dienoic
acid (6)
NaOH (4.8 g) was added to a suspension of compound 5
(18 g) in t-BuOH (200 ml) and the whole was next heated
until reflux began. This was maintained for 1 h, following
which compound 6 (10.1 g, 69%) was obtained after purifi-
cation through dry column flash chromatography on silica
gel, eluting with EtOAc:cyclohexane 1:1. Amorphous pow-
der. UV (CH2Cl2)kmax (nm) (loge): 265 (4.3). FTIR (KBr)
3.8.4. Synthesis of (2S,3S)-3-methyl-2-[(20E,40E)-20-
methylhexa-20,40-dienoylamino]- pentanoic acid ethyl ester
(8)
The procedure was similar as above. N-Boc-L-Ile methyl
ester (400 mg) was employed instead of the D-allo-Ile deriv-
ative. After purification, compound 8 (200 mg) was
obtained. Compound 8: Oil. [a]D = +9 (CHCl3, c2.72).
UV (CH2Cl2)kmax (nm) (loge): 244 (4.4). FTIR (KBr) mmax
(cmꢀ1): 3320 (NH), 2970 (CH), 2929 (CH), 2860 (CH),
1750 (CO), 1631, 1372, 697. 1H NMR (CDCl3): d 6.88
(br d, J = 11.1 Hz, H-30), 6.33 (ddq, J = 14.9, 11.1 and
1.6 Hz, H-40), 6.24 (br d, J = 8.4 Hz, NH), 6.04 (dq,
J = 14.9 and 7.0 Hz, H-50), 4.68 (dd, J = 8.4 and 5.0 Hz,
H-2), 3.75 (s, CO2CH3), 1.96 (br s, H-70), 1.94 (m, H-3),
1.86 (br d, J = 7.0 Hz, H-60), 1.48 and 1.21 (m, H-4), 0.94
(t, J = 7.4 Hz, H-5), 0.92 (d, J = 7.0 Hz, H-6). 13C NMR
(CDCl3): d 172.8 (s, C-1), 168.9 (s, C-10), 136.4 (d, C-50),
134.3 (d, C-30), 127.3 (s, C-20), 127.0 (d, C-40), 56.5 (d, C-
2), 52.0 (s, CO2CH3), 38.2 (d, C-3), 25.4 (t, C-4), 18.8 (q,
C-60), 15.4 (q, C-6), 12.8 (q, C-70), 11.6 (q, C-5). HREIMS
m/z: (M)+Æ, found 253.16899, calc. for C14H23NO3
253.16779 (Dm = 4.7 ppm), (M + 1)+Æ found 254.17115,
calc. for 13CC13H23NO3 254.17118 (Dm = 1.1 ppm). EIMS
70 eV, m/z (rel. int.): 253 [M]+Æ(9), 238 (4), 224 (4), 144 (14),
128 (12), 109 [RCO]+ (100), 81 (25).
m
max (cmꢀ1): 2935 (CH), 2656, 2595, 1689 (CO), 1640, 1612,
1441, 1305, 1270, 969, 940. 1H NMR (CDCl3): d 7.27 (br d,
J = 11.4 Hz, H-3), 6.38 (ddq, J = 15.1, 11.4 and 1.8 Hz, H-
4), 6.15 (dq, J = 15.1 and 6.7 Hz, H-5), 1.91 (br s, H-7),
1.89 (br d, J = 6.7 Hz, H-6). 13C NMR (CDCl3) : d 173.9
(s, C-1), 140.9 (d, C-3), 139.1 (d, C-5), 127.4 (d, C-4),
123.9 (s, C-2), 18.9 (q, C-6), 12.1 (q, C-7). EIMS 70 eV,
m/z (rel. int.): 126 [M]+Æ(37), 111 (81), 79 (44), 55 (47), 43
(80), 41 (100).
3.8.3. Synthesis of (2R,3S)-3-methyl-2-[(20E,40E)-20-
methylhexa-20,40-dienoylamino]- pentanoic acid ethyl ester
(7)
N-Boc-D-allo-Ile methyl ester was prepared according to
the procedure of Hamada et al. (1987). Dry EtOAc satu-
rated with HCl (vapor) (5 ml) was added to a solution of
N-Boc-D-allo-Ile methyl ester (340 mg) in dry EtOAc
(5 ml). After 5 h of stirring at room temperature, the crude

G.L. Gallardo et al. / Phytochemistry 67 (2006) 2403–2410
2409
3.8.5. Synthesis of compound 1
J = 8.0 Hz, NH), 4.29 (m, H-2), 1.70 (m, H-3), 1.47 (over-
lapped, H-4), 1.45 (s, C(CH3)3), 1.27 (m, H-4), 0.99 (d,
J = 6.9 Hz, H-6), 0.96 (t, J = 7.3 Hz, H-5). 13C NMR
(CDCl3): d 200.6 (d, C-1), 156.9 (s, NCOO), 79.6 (s,
C(CH3)3) 64.2 (d, C-2), 36.4 (d, C-3), 28.3 (s, C(CH3)3),
25.3 (t, C-4), 15.6 (q, C-6), 11.9 (q, C-5). HREIMS m/z:
(M)+Æ, found 215.15258, calc. for C14H23NO3 215.15214
(Dm = 2 ppm). EIMS m/z (rel. int.): 215 [M]+Æ (50), 186
(27), 130 (88), 86 (79), 57 (100).
LiBH4 (14 mg) and MeOH (0.025 ml) were added to a
solution of compound 7 (70 mg) in Et2O (2 ml). The mix-
ture was heated until reflux began, this being maintained
for 15 min, with the resulting reaction mixture poured onto
1 N HCl in an ice-bath. The reaction mixture was next
extracted with CH2Cl2 (3 · 25 ml). The crude evaporated
product was subjected to dry column flash chromatogra-
phy on silica gel, eluting pure compound 1 (30 mg) with
CH2Cl2:EtOAc 70:30.
Compound 1. Oil. [a]D = ꢀ45 (CHCl3, c0.1). UV
(CH2Cl2)kmax (nm) (log e): 236 (3.0). FTIR (KBr) mmax
(cmꢀ1): 3321 (NH), 2932, 2846, 1742, 1489, 1380, 1193.
1H NMR (CDCl3): d 6.90 (br d, J = 10.8 Hz, H-3), 6.33
(ddq, J = 14.8, 10.8 and 1.6 Hz, H-4), 6.04 (dq, J = 14.8
and 6.9 Hz, H-5), 5.90 (br d, J = 8.0 Hz, NH), 4.00 (m,
H-20), 3.69 (m, H-10), 1.96 (br s, H-7), 1.86 (br d,
J = 6.9 Hz, H-6), 1.70 (m, H-30), 1.44 and 1.25 (m, H-40),
0.93 (t, J = 7.3 Hz, H-50), 0.93 (d, J = 6.9 Hz, H-60). 13C
NMR (CDCl3): d 170.3 (s, C-1), 136.3 (d, C-5), 134.2 (d,
C-3), 127.3 (s, C-2), 126.9 (d, C-4), 64.3 (t, C-10), 55.3
(d, C-20), 35.5 (d, C-30), 26.3 (t, C-40), 18.7 (q, C-6), 14.8
(q, C-60), 12.8 (q, C-7), 11.3 (q, C-50). HREIMS m/z:
(M)+Æ, found 225.17263, calc. for C14H23NO3 225.17288
(Dm = 1.1 ppm). EIMS 70 eV, m/z (rel. int.): 225
[M]+Æ(8), 194 (22), 109 [RCO]+ (100), 86 (62).
3.8.8. Antibiotic activity
The antibiotic activity was determined by the agar diffu-
sion method using 50 lg of sample/6 mm disk against B.
subtilis ATCC 6633, S. aureus ATCC 25923 and E. coli
ATCC 25922. Gentamicin, which was used as the positive
test compound, showed inhibition halos of 25–30 mm at
a conc. level of 25 lg/disk.
3.8.9. Antifungal activity
Direct bioautography on TLC was employed as the
method for detecting fungitoxic substances (Homans and
Fuchs, 1970). A concentration level of 50 lg/spot of each
assayed compound was used. Benomyl, which was used
as a control, showed inhibition zones of 20–28 mm at a
conc. level of 25 lg/spot. When appropriate, as large inhib-
itory halos were observed, minimum inhibitory concentra-
tion (MIC) was measured by the same method.
3.8.6. Synthesis of compound 2
The procedure was similar as above. Compound 8
(150 mg) was employed instead of 7. After purification,
pure compound 2 (80 mg) was obtained. Compound 2: Oil.
[a]D = +72 (CH2Cl2, c0.37). UV (CH2Cl2)kmax (nm) (loge):
240 (4.0). FTIR (KBr) mmax (cmꢀ1): 3390 (NH), 2968, 2939,
1662, 1539, 1467, 1388, 1085. 1H NMR (CDCl3): d 6.88 (br
d, J = 10.8 Hz, H-3), 6.33 (ddq, J = 14.8, 10.8 and 1.5 Hz,
H-4), 6.03 (dq, J = 14.8 and 6.9 Hz, H-5), 6.00 (overlapped,
NH), 3.88 (m, H-20), 3.73 (m, H-10), 1.95 (br s, H-7), 1.86 (br
d, J = 6.9 Hz, H-6), 1.70 (m, H-30), 1.50 and 1.22 (m, H-40),
0.95 (d, J = 6.8 Hz, H-60), 0.91 (t, J = 7.0 Hz, H-50). 13C
NMR (CDCl3): d 170.0 (s, C-1), 136.4 (d, C-5), 134.2 (d,
C-3), 127.2 (s, C-2), 127.0 (d, C-4), 63.7 (t, C-10), 56.2
(d, C-20), 35.7 (d, C-30), 25.4 (t, C-40), 18.7 (q, C-6), 15.5
(q, C-60), 12.7 (q, C-7), 11.6 (q, C-50). HREIMS m/z:
(M)+Æ, found 225.17197, calc. for C14H23NO3 225.17288
(Dm = 4.0 ppm). EIMS m/z (rel. int.): 225 [M]+Æ (5), 210
(1), 195 (3), 194 (12), 126 (11), 109 [RCO]+ (100), 86 (20),
81 (28).
Acknowledgements
We thank UMYMFOR (CONICET-FCEN, UBA) for
the NMR and LR-MS spectra and Universidad de Buenos
Aires, ANPCYT and CONICET for partial financial
support.
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