Insect toxins from spruce endophytes

Article abstract of DOI:10.1139/v03-044

Extracts of fermentation cultures of a fungal endophyte (DAOM 221611) from spruce needles have afforded the known macrocyclic antibiotic vermiculin (1), 7α,8β,11-trihydroxydrimane (2), and eight novel 13-carbon γ-lactones, namely trans-3-methyldodec-cis-6-en-4-olide (3), trans-8-hydroxy-3-methyldodec-cis-6-en-4-olide (4), trans-8-acetoxy-3-methyldodec-cis-6-en-4-olide (5), trans-9-hydroxy-3-methyl-8-oxo-dodec-trans-6-en-4-olide (6), trans-8,9-dihydroxy-3-methyldodec-cis-6-en-4-olide (7), trans-9-hydroxy-8-oxo-3-methyldodecan-4-olide (8), trans-7,9-dihydroxy-3-methyl-8-oxo-dodecan-4-olide (9), and trans-6-hydroxymethyl-3-methyl-7-oxo-undecan-4-olide (10). A known JH III metabolism product, 10,11-dihydroxyfarnesenic acid (11), was also isolated and synthesized from farnesol. Other endophyte cultures from black spruce and white spruce afforded the novel 6,7-dihydroxy-2-propyl-2,4-octadien-4-olide (16), 5,6,8-trihydroxy-4-(1′-hydroxyethyl) isocoumarin (17) plus the known sescandelin (18), sescandelin B (19), and 4-hydroxy-2-methoxyacetanilide (20). Several of the γ-lactones showed toxicity to spruce budworm (Choristoneura fumiferana Clem.) larvae and vermiculin 1 and compound 16 were toxic to spruce budworm cells.

Full text of DOI:10.1139/v03-044

284
Insect toxins from spruce endophytes
John A. Findlay, Guoqiang Li, J. David Miller, and Taiwo O. Womiloju
Abstract: Extracts of fermentation cultures of a fungal endophyte (DAOM 221611) from spruce needles have afforded
the known macrocyclic antibiotic vermiculin (1), 7α,8β,11-trihydroxydrimane (2), and eight novel 13-carbon γ-lactones,
namely trans-3-methyldodec-cis-6-en-4-olide (3), trans-8-hydroxy-3-methyldodec-cis-6-en-4-olide (4), trans-8-acetoxy-3-
methyldodec-cis-6-en-4-olide (5), trans-9-hydroxy-3-methyl-8-oxo-dodec-trans-6-en-4-olide (6), trans-8,9-dihydroxy-3-
methyldodec-cis-6-en-4-olide (7), trans-9-hydroxy-8-oxo-3-methyldodecan-4-olide (8), trans-7,9-dihydroxy-3-methyl-8-
oxo-dodecan-4-olide (9), and trans-6-hydroxymethyl-3-methyl-7-oxo-undecan-4-olide (10). A known JH III metabolism
product, 10,11-dihydroxyfarnesenic acid (11), was also isolated and synthesized from farnesol. Other endophyte cul-
tures from black spruce and white spruce afforded the novel 6,7-dihydroxy-2-propyl-2,4-octadien-4-olide (16), 5,6,8-
trihydroxy-4-(1′-hydroxyethyl) isocoumarin (17) plus the known sescandelin (18), sescandelin B (19), and 4-hydroxy-2-
methoxyacetanilide (20). Several of the γ-lactones showed toxicity to spruce budworm (Choristoneura fumiferana
Clem.) larvae and vermiculin 1 and compound 16 were toxic to spruce budworm cells.
Key words: toxigenic endophytes, insect toxins, γ-lactones, isocoumarins.
Résumé : L’extraction des milieux de cultures de fermentation d’un champignon endophyte (DAOM 221611) des ai-
guilles d’épinette a permis d’isoler la vermiculine (1), un antibiotique macrocyclique connu, le 7α,8β,11-trihydroxy-
drimane (2) ainsi que huit nouvelles γ-lactones à 13 atomes de carbone, soit les trans-3-méthyldodéc-cis-6-én-4-olide
(3), trans-8-hydroxy-3-méthyldodéc-cis-6-én-4-olide (4), trans-8-acétoxy-3-méthyldodéc-cis-6-én-4-olide (5), trans-9-
hydroxy-3-méthyl-8-oxododéc-cis-6-én-4-olide (6), trans-8,9-dihydroxy-3-méthyldodéc-cis-6-én-4-olide (7), trans-9-
hydroxy-8-oxo-3-méthyldodécan-4-olide (8), trans-7,9-dihydroxy-3-méthyl-8-oxododécan-4-olide (9) et trans-6-hydroxy-
méthyl-3-méthyl-7-oxoundécan-4-olide (10). On a aussi isolé un produit connu de métabolisme JH III, l’acide 10,11-
dihydroxyfarnésique (11) qu’on a synthétisé à partir du farnésol. D’autres milieux de cultures d’endophytes provenant
d’épinettes noire ou blanche ont permis d’isoler de nouveaux produits, dont le 6,7-dihydroxy-2-propylocta-2,4-dién-4-
olide (16) ainsi que la 5,6,8-trihydroxy-4-(1′-hydroxyéthyl)isocoumarine (17) en plus de produits connus comme la ses-
candéline (18), la sescandéline B (19) et le 4-hydroxy-2-méthoxyacétanilide (20). Plusieurs γ-lactones présentent de la
toxicité pour les larves de la tordeuse des bourgeons d’épinette (Choristoneura fumiferana Clem.) alors que la vermicu-
line 1 et le composé 16 sont toxiques pour les cellules de la tordeuse des bourgeons d’épinette.
Mots clés : endophytes toxigènes, toxines d’insectes, γ-lactones, isocoumarines.
[Traduit par la Rédaction] Findlay et al. 292
Recently, we reported that inoculation of white spruce,
cultures capable of producing an interesting array of novel
γ-lactones (3–10), as well as 10,11-dihydroxyfarnesenic acid
11, the macrocyclic antibiotic, vermiculin 1 (4), and the
known sesquiterpene, 7α,8β,11-trihydroxydrimane 2 (5).
Other spruce-needle-derived endophyte cultures gave the
novel 2,4-dien-4-olide 16, the novel isocoumarin 17, plus
the known sescandelin 18 (6), sescandelin B 19 (7), and 4-
hydroxy-2-methoxyacetanilide 20 (Scheme 1). Both 18 and
19 have been previously isolated from the fungus Sesquil-
lium candelabrum, and sescandelin 18 is reported to be a
root-promoting agent (6). Compound 20 has recently been
reported as a metabolite of a soil fungus (8).
Picea glauca (Moench voss.), seedlings with a rugulosin-
producing endophyte resulted in a significant reduction in
larval growth rate of the destructive forest pest, the spruce
budworm Choristoneura fumiferana Clem., in feeding exper-
iments (1). This latter study followed from our pioneering
discovery that some conifer needles are inhabited by insect-
toxin-producing endophytes (2), consistent with the proposal
of Carroll and Carroll that fungal endophytes recovered from
conifer needles might be mutualistic symbionts (3). In our
continuing studies in search of additional toxin-producing
endophytic fungi from spruce needles we have encountered
The endophyte strain 5WS11I, obtained from needles of a
white spruce (P. glauca (Moench voss)), was cultured ac-
cording to established protocols (9) using two different kinds
of vessels. In the first case the fermentation was conducted
in 250-mL Erlenmeyer flasks (2 L total), and in the second
case Glaxo bottles (10 L total) were employed but no other
changes in culturing procedures were made, and the fermen-
tations proceeded simultaneously. The fermentation-medium
filtrates were extracted with EtOAc, and crude extracts E
(from Erlenmeyer flask culture) and G (from Glaxo bottle
culture) were obtained by removal of solvent under vacuum
Received 12 December 2002. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 28 April 2003.
J.A. Findlay1 and G. Li. Department of Chemistry,
University of New Brunswick, Fredericton, NB E3B 6E2,
Canada.
J.D. Miller and T.O. Womiloju. Ottawa-Carleton Institute of
Chemistry, Department of Chemistry, Carleton University,
Ottawa, ON K1S 5B6, Canada.
1Corresponding author (e-mail: findlay@unb.ca)
Can. J. Chem. 81: 284–292 (2003)
doi: 10.1139/V03-044
© 2003 NRC Canada

Findlay et al.
285
Scheme 1.
at room temperature. From the crude extract G, a number of
γ-lactones 3–10 and the sesquiterpenes 11 and 7α,8β,11-
trihydroxydrimane have been isolated and their structures
elucidated by spectral data analyses. From the crude extract
E, only the 16-membered ring dilactone, vermiculin, and
7α,8β,11-trihydroxydrimane were isolated. The antibiotic
vermiculin was previously reported from Penicillium
vermiculatum (4) and a soil fungus Talaromyces wortmannii
(10) while the γ-lactones 3–10 are closely related to a γ-
decalactone (11) and cis-6-dodecen-4-olide (12), fungal me-
tabolites previously reported from the cultures of Sporobolo-
myces odorus. Sesquiterpene 11 was previously reported as
a biotransformation product of the corresponding 10,11-
epoxide, an insect juvenile hormone (JHIII) homologue (13),
and 7α,8β,11-trihydroxydrimane was isolated previously
from the fungus, Fomes annosus, a destructive forest patho-
gen (5). Compounds 4, 6–8, 11 and 7α,8β,11-trihydroxy-
drimane 2 were toxic to spruce budworm in a larval feeding
bioassay, and vermiculin 1 and 16 were toxic to budworm
cells.
Compound 3 was isolated as an optically active, oily sub-
stance from a nonpolar fraction of the crude extract G. Its
molecular formula, C13H22O2, was determined by HR-EI-
MS. The IR spectrum is consistent with the presence of a
lactone carbonyl group (1780 cm–1) and a vinyl group
(1640 cm–1
), and the 13C NMR and DEPT spectra (Table 1)
indicate the presence of one carbonyl, four methines, six
methylenes, and two methyls. A total of four downfield car-
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Findlay et al.
287
bon signals are assigned to an ester carbonyl, a double bond,
and an oxygenated methine carbon. The proton and carbon
[M+ – C4H8O] and [M+ – C4H9O] correspond to cleavage
1
at the C-8 — C-9 bond. The H-1H COSY spectrum dis-
1
chemical shifts were assigned on the basis of analysis of H
plays
two
distinct
coupled
spin
systems,
1
NMR, 13C NMR, DEPT, HMQC, H-1H COSY, and HMBC
CH3CH2CH2CHO- and -CH=CHCH2CHCH(Me)CH2-, es-
tablishing the connectivity and substitution pattern in 6. The
coupling constant (J = 15.6 Hz) for H-6/H-7 indicates a
trans double bond, and HMBC correlations of H-6 and H-9
to C-8 and of H-2 and H-4 to C-1 complete the structural
analysis, and we conclude that 6 is trans-9-hydroxy-3-
methyl-8-oxo-dodec-trans-6-en-4-olide.
Compound 7 was isolated as an optically active, oily sub-
stance whose IR spectrum is nearly identical to that of γ-
lactone 4 and indicates the presence of hydroxyl (3430 cm–1),
vinyl (1630 cm–1), and lactone (1775 cm–1) functions. The
13C NMR and DEPT spectra display one additional oxygen-
ated methine relative to 4. The molecular formula was deter-
mined as C13H22O4 on the basis of MS and NMR data. The
1H-1H COSY spectrum revealed an extended spin-coupling
system containing the sequence -CH2CHCHCH=CHCH2CH-
CH(Me)CH2-, and the coupling constant (J = 11.2Hz) for H-
6/H-7 established the cis geometry of the double bond. Thus
lactone 7 is trans-8,9-dihydroxy-3-methyldodec-cis-6-en-4-
olide.
spectra and NOE data, as were those of the other compounds
1
reported here. The H-1H COSY spectrum displays a cou-
pled spin system, which fully supports the sequence of pro-
tons from C-2 to C-8 and the methyl at C-3. The location of
the γ-lactone is apparent from the chemical shifts of H-2 and
H-4 and confirmed by HMBC correlations from the protons
at C-2 to the C-1 carbonyl. The trans relationship of the two
substitutents on the γ-lactone ring is concluded on the basis
of NOE enhancements between H-4 and methyl-13. The
double bond is assigned the cis geometry based on the cou-
pling constant (J = 11.0 Hz) between H-6 and H-7 and the
large NOE enhancements between them. We therefore as-
sign the structure trans-3-methyldodec-cis-6-en-4-olide to 3.
The optically active, oily γ-lactone alcohol 4 was the ma-
jor metabolite isolated from the extract G. Its IR spectrum
supports the presence of hydroxyl (3443 cm–1) and lactone
(1777 cm–1) functional groups. The 13C NMR and DEPT
spectra are very similar to those of γ-lactone 3 but feature
one additional oxygenated methine in place of one methy-
lene carbon. The molecular formula, C13H22O3, was deduced
on the basis of NMR spectral data and supported by an HR-
EI-MS (found: m/z = 226.1584; calculated for C13H22O3:
Compound 8 was isolated as an optically active, oily
substance whose IR spectrum indicates the presence of
hydroxyl (3474 cm–1), lactone (1777 cm–1), and carbonyl
(1711 cm–1) functionalities. Its molecular formula was de-
termined as C13H22O4 on the basis of NMR and HR-EI-MS
1
226.1569, [M+]) The H-1H COSY spectrum shows an ex-
tended coupled spin system, similar to that of compound 3,
supporting the C-2 to C-8 substitution pattern. The γ-lactone
ring is established by comparing the 13C chemical shifts with
those of compound 3 and confirmed in detail by HMBC cor-
relations. The relative stereochemistry of the five-membered
ring lactone was resolved by the NOE enhancements be-
tween the methine proton at H-4 and methyl-13. The cis ge-
ometry of the double bond was apparent from the NOE
enhancements between the homo-allylic protons H-8 and H-
5, and thus we conclude 4 is trans-8-hydroxy-3-methyl-
dodec-cis-6-en-4-olide.
1
data (Table 2). The H-1H COSY spectrum shows the two
independent spin coupling systems CH3CH2CH2CHO- and
-CH2CH2CH2CHCH(Me)CH2- in agreement with formula-
tion 8. The HMBC correlations of H-7 and H-9 with C-8
and H-2 and H-4 with C-1 and NOE enhancements between
H-4 and Me-13 support the conclusion that 8 is trans-9-
hydroxy-3-methyl-8-oxo-dodecan-4-olide.
The minor compound 9 was isolated as an optically active
oil and its IR spectrum indicates the presence of hydroxyl
(3417 cm–1) and carbonyl (1777 and 1757 cm–1) functional
groups. The 13C NMR and DEPT spectra are similar to those
of γ-lactone 8 but feature an additional oxygenated methine
in place of a methylene. The molecular formula, C13H22O5,
is assigned based on spectral data, including an EI-MS frag-
mentation pattern paralleling that of 6 with loss of C4H9O
from the molecular ion due to cleavage at the C-8 — C-9
bond. The placement of the second oxygenated methine at
The IR spectrum of optically active oil 5 shows the pres-
ence of vinyl (1641 cm–1), ester (1729 cm–1), and lactone
1
(1781 cm–1) functional groups. The H NMR spectrum is
nearly identical to that of γ-lactone alcohol 4 except for the
presence of an additional methyl singlet at δ 2.01 and the
downfield shift of H-8, indicating the presence of an acetate
group. The mass spectral data support a molecular formula
of C15H24O4 and show a major fragment corresponding to
1
C-7 follows from the H-1H COSY spectrum, which affirms
1
[M+ – C2H2O], typical for acetates. The H-1H COSY spec-
the presence of the -CH(O)CH2CH2CH(O)CH(Me)CH2-
moiety. HMBC correlations and NOE data confirm our con-
trum displays an extended coupling sequence, attesting to
the C-2 to C-8 substitution pattern, and the stereochemistry
of the double bond and lactone ring substituents were re-
solved by NOE and coupling constant data as in the case of
3. We therefore conclude that 5 is trans-8-acetoxy-3-methyl-
dodec-cis-6-en-4-olide.
The IR spectrum of the optically active oil 6 indicates the
presence of hydroxyl (3459 cm–1), vinyl (1632 cm–1), ketone
(1713 cm–1), and lactone (1777 cm–1) functional groups. Its
13C NMR spectrum and DEPT spectra are similar to those of
compound 3 but feature one more carbonyl in place of a
methylene carbon. The molecular formula, C13H20O4, was
determined on the basis of NMR data. The HR-EI-MS spec-
trum did not show a molecular ion, but major fragments of
clusion that
9
is trans-7,9-dihydroxy-3-methyl-8-oxo-
dodecan-4-olide.
The IR spectrum of the optically active, oily γ-lactone 10
indicates the presence of hydroxyl (3471 cm–1) and carbonyl
(1778, 1723 cm–1) groups. The 13C NMR and DEPT spectra
show a total of 13 carbons, comprising two methyls, six
methylenes, three methines, and two quaternary carbons.
The composition, C13H22O4, was ascertained from EI-MS
and NMR data. The two discreet spin coupling systems,
CH3CH2CH2CH2- and CH2(O)CHCH2CHCH(Me)CH2-, are
1
apparent from the H-1H COSY spectrum. Comparison of
spectral data with those for the similar compounds 8 and 9
led us to conclude that 10 is trans-6-hydroxymethyl-3-
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Can. J. Chem. Vol. 81, 2003
Table 2. NMR data (CDCl3) for lactones 8–10.
Compound 8
Compound 9
Compound 10
Carbon
δ
Proton (J Hz)
δ
Proton (J Hz)
δ
Proton (J Hz)
1
2
3
4
5
6
7
8
176.2 (s)
36.9 (t)
36.1 (d)
87.0 (d)
33.2 (t)
19.9 (t)
37.2 (t)
211.8 (s)
176.2 (s)
37.0 (t)
36.4 (d)
86.9 (d)
29.9 (t)
31.0 (t)
73.3 (d)
213.5 (s)
177.2 (s)
2.67 (m, β), 2.19 (m, α) 36.9 (t)
2.65 (m, β), 2.16 (m, α)
2.19 (m, β)
3.97 (ddd, α, 10.8, 8.8, 3.2)
1.76 (m), 1.56 (m)
1.81 (m), 1.76 (m)
2.53 (t, 7.2), 2.51 (t, 6.8)
2.65 (m, β), 2.18 (m, α)
2.19 (m, β)
3.87 (ddd, α, 10.0, 7.2, 2.0)
2.24 (m), 1.59 (m)
3.04 (m)
2.20 (m, β)
3.98 (m, α)
1.96 (m), 1.69 (m)
2.09 (m), 1.55 (m)
4.43 (m)
37.0 (d)
85.2 (d)
32.8 (t)
50.4 (d)
213.3 (s)
43.5 (t)
2.57 (dt, 17.2, 7.6),2.53 (dt,
17.2, 7.2)
9
76.2 (d)
35.9 (t)
18.2 (t)
13.9 (q)
4.15 (ddd, 8.0, 4.8, 4.4)
1.75 (m), 1.47 (m)
1.48 (m), 1.37 (m)
0.93 (3H, t, 7.3)
73.6 (d)
36.4 (t)
18.1 (t)
13.8 (q)
4.45 (m)
25.4 (t)
22.3 (t)
13.9 (q)
64.0 (t)
1.54 (2H, m)
1.31 (2H, m)
0.89 (3H, t, 7.3)
3.81 (dd, 11.2, 4.8),3.75
(dd, 11.2, 5.2)
10
11
12
1.76 (m), 1.51 (m)
1.49 (m), 1.42 (m)
0.96 (3H, t, 7.2)
13
17.3 (q)
1.12 (3H, d, 6.3)
17.1 (q)
1.14 (3H, d, 6.4)
16.9 (q)
1.12 (3H, d, 6.2)
methyl-8-oxo-undecane-4-olide. This conclusion is fully
consistent with HMBC and NOE experiments.
the presence of an n-propyl group and the sequence CH3CH-
CHCH- assigned to the side chains H-3′, H-2′, H-1′ and H-8,
H-7, H-6, H-5, respectively, in structure 16. Furthermore,
the signal for H-5 (5.34 ppm) shows a long-range coupling
to the other vinylic proton H-3 (δ 7.37), while both of these
show further couplings to the signal for H-1′ (δ 2.31) consis-
tent with their relationships in structure 16. The placement
of the lactone carbonyl follows from the HMBC correlations
from H-1′ and H-3 to C-1.The NOE difference spectrum dis-
plays significant correlations from H-3 to H-1′ and H-5. The
UV spectrum (λmax = 272) is close to the calculated value
(λmax = 268) for this dienone chromophore. The HR-EI-MS
showed an [M+ – H2O] ion at m/z 194.0962 (calcd. 194.0943
for C11H14O3), which supports formulation 16. Thus, we
conclude compound 16 is 6,7-dihydroxy-2-propyl-octa-2,4-
dien-4-olide. A conformational search of 16 (in acetone) re-
vealed that four out of five of the lowest-energy conforma-
tions for the threo isomer displayed an H-6/H-7 dihedral
angle near to 180°, whereas the erythro lowest-energy con-
formations favoured an H-5/H-6 dihedral angle near 70°,
consistent with the observed coupling constant of 4.3 Hz.
2,4-Dien-4-olides have been reported from the gorgonians
Plexaura flava (14) and Euplexaura flava (15) and from the
liverwort Marchantia paleacea subsp. diptera (16) but not
previously from a fungal source.
Compound 11 was isolated as an optically active oil
whose IR spectrum supports the presence of hydroxyl, α,β-
unsaturated carboxyl (3500–2500, 1694 cm–1), and vinyl
(1647 cm–1) groups. The composition, C15H26O4, follows
from NMR spectral data and an HR-EI-MS that lacks a mo-
lecular ion but features prominent ions corresponding to
[M+ – C3H7O] and [M+ – C3H7O – H2O]. The 13C NMR and
DEPT data attest to the presence of four methyls, four
methylenes, two sp2 methines and one sp3 methine, three
fully substituted sp2 carbons, and one quaternary carbon.
The 1H-1H COSY spectrum shows the two separate coupling
spin systems =CHCH2CH2- and -CH(O)CH2CH2-. The con-
nection of these and the remaining structural units is readily
achieved by an examination of the HMBC correlations
(Fig. 1). The strong NOE correlations of H-2 with H-4 and
H-6 with H-8 indicate the trans stereochemistry of the dou-
ble bonds, and we conclude that 11 is trans,trans-10,11-
dihydroxyfarnesenic acid.
It should be noted that 11 is closely related to a Cecropia
juvenile hormone (JHIII), methyl 10,11-epoxy-3,7,11-trim-
ethyldodecanoate 15, which profoundly affects metamorpho-
ses of many insects and that 11 has been found to be a
metabolic product of JHIII (13). The structure of this
sesquiterpene was confirmed via synthesis. Farnesol was
converted to farnesenic acid via a two-step oxidation by
MnO2 followed by NaClO2–NaH2PO4. The resulting acid
was treated with N-bromosuccinimide, producing the corre-
sponding 10,11-bromohydrin 12 in 74% isolated yield,
which was then converted to the corresponding 10,11-
epoxide 13. Opening the epoxide ring under acidic condi-
tions produced the racemic diol 11 in 46% yield from 12
plus the 11-membered lactone 14 in 36% yield from 12.
This lactone can be converted to the racemic diol 11 upon
treatment with base.
Compound 17 was isolated as an optically active, yellow-
ish solid, and its molecular formula C11H10O6 was deter-
mined by its HR-EI-MS molecular ion at m/z 238.0467
1
(calcd. 238.0477). The H NMR spectrum of 17 (Table 4)
closely resembles that of sescandelin 18 except for a signal
corresponding to H-5. The 13C NMR data attest to the pres-
ence of nine sp2 carbons, two of which are protonated, plus
a methine and a methyl group. There is a close correspon-
dence in the chemical shift of signals for 17 assigned to C-1,
C-3, C-4, C-8a, C-1′, and C-2′ and those of sescandelin 18
(6). The HMBC correlations of the signal at δ 11.47 with
signals assigned to C-7, C-8, and C-8a establish the 5,6,8-
substitution pattern of the aromatic ring, and correlations be-
tween the aromatic proton signal at δ 6.56 with signals as-
signed to C-5, C-6, C-8, and C-8a confirm this conclusion.
The 13C NMR data (Table 3) of compound 16 support the
presence of five sp2 carbons, two of which are protonated,
together with two methyl, two methylene, and two oxygen-
1
substituted methines. The H-1H COSY spectrum attests to
© 2003 NRC Canada

Findlay et al.
289
Fig. 1. Selected HMBC correlations for 11.
Table 3. NMR data (acetone-d6) for compound 16.
Carbon
δ
Proton (J Hz)
HMBC (H to C)
NOE diff. (%)
1
2
3
4
5
6
7
8
1′
2′
3′
170.8
135.1
138.7
150.0
114.0
71.3
71.0
19.0
27.9
21.7
7.37 (s)
C-1, C-4, C-1′
H-5 (3.1), H-1′ (0.7), H-2′ (1.0)
5.34 (d, 9.0)
4.52 (dd, 9.1, 4.3)
3.83 (m)
1.09 (d, 6.7)
2.31 (m)
C-3, C-4, C-6, C-7
C-4, C-7, C-8
C-5, C-6
H-3 (4.0), H-6 (0.3), H-7 (0.8)
H-5 (0.8), H-7 (1.1), H-8 (0.7)
H-5 (0.2), H-6 (1.9), H-8 (3.1)
H-5 (0.5), H-6 (1.2), H-7 (1.7)
H-3 (0.8), H-2′ (2.6)
C-6, C-7
C-1, C-2, C-3, C-2′, C-3′
C-2, C-1′, C-3′
C-1′, C-2′
1.60 (m)
0.95 (t,7.5)
H-3 (1.2), H-1′ (2.1), H-3′ (3.2)
H-1′ (0.8), H-2′ (3.3)
14.1
Table 4. NMR data (acetone-d6) for compound 17.
Carbon
δ
Proton (J Hz)
HMBC (H to C)
NOE diff. (%)
1
3
4
4a
5
6
7
8
166.2
140.1
119.5
119.6
132.1
155.9
102.7
158.6
97.8
7.36 (s)
C-1, C-4, C-1′
H-2′ (0.7), H-1′ (2.9)
6.56 (s)
C-5, C-6, C-8, C-8a
8a
1′
2′
8-OH
1′-OH
67.2
22.1
5.02 (q, 6.7)
1.54 (d, 6.7)
11.47 (s)
6.05 (bs)a
C-3, C-4, C-2′
C-4, C-1′
C-7, C-8, C-8a
H-2′ (4.0), H-3 (3.9)
H-1′ (1.1), H-3 (0.4)
aMeasured in DMSO-d6.
The 13C NMR chemical shifts of C-4a, C-6, and C-8 in 17
are significantly downfield relative to the corresponding sig-
nals of sescandelin 18, consistent with the presence of an
hydroxyl at C-5. The absolute configuration of 17 is implied
from the co-occurrence of S-(–)-6,8-dihydroxy-4-(1′-hydr-
oxyethyl) isocoumarin 18 with the same sign of optical rota-
tion. Isocoumarin derivatives are fairly common metabolites
of plants, fungi, and insects and show a wide range of bio-
activities (14). While 6,8-dihydroxylated variations are quite
common, compound 17 is the first example of a 5,6,8-trihy-
droxyisocoumarin. Two 6,8-dihydroxy-5-methoxyisocou-
marin derivatives have been reported from both Aspergillus
ustus (17) and Paecilmyces victoriae (18).
20–30% as toxic as the control, deoxynivalenol, while 17,
18, and 19 were individually weakly toxic. These findings
may provide additional options for our ongoing development
of insect resistance in conifers via inoculation of seedlings
with toxin-producing endophytes (1).
Experimental section
General
High-resolution mass spectra (HR-EI-MS) were recorded
on a Kratos MS-50 instrument. Optical rotations were mea-
sured on a PerkinElmer 241 polarimeter. Melting points
were taken with a Kofler hot-stage apparatus and are uncor-
rected. The IR spectra were recorded as films on a Bruker
IF-S25 spectrometer. UV spectra were determined on a
PerkinElmer 330 spectrometer. Preparative thin layer chro-
Crude extracts and compounds 16–19 were tested for
cytotoxicity against the spruce budworm (C. fumiferana
Clem.) cell line CF-1 (9). Compound 16 was found to be
© 2003 NRC Canada

290
Can. J. Chem. Vol. 81, 2003
matography (ptlc) was performed with precoated silica gel
F254 (1 mm) plates unless otherwise indicated. NMR data
were recorded at room temperature in CDCl3 (unless other-
wise specified) on a Varian Unity 400 spectrometer operat-
1157 (s), 1007 (s), 920 (s), 733 (s). HR-EI-MS m/z (%):
226.1584 ([M+], 100) (calcd. for C13H22O3: 226.1569). EI-
MS m/z (%): 226 ([M]+, 9.0), 209 (13.6), 208 (8.8), 197
(11.1), 184 (46.3), 170 (18.7), 169 (75.6), 167 (15.0), 166
(35.8), 154 (24.7), 152 (55.7), 151 (100), 139 (66.1), 127
(40.3), 110 (83.3), 109 (85.7), 105 (54.0), 99 (98.1), 95
(77.6), 85 (72.9), 81 (70.9), 71 (75.1).
1
ing at 399.944 MHz for H and 100.575 MHz for 13C. All
2D NMR were recorded non-spinning at 25°C using stan-
dard Varian software (VNMR 4.3).
γ-Lactone 5
Fungal strain 5WS11I1 (DAOM 221611)
An oil, [α]D = +12.8° (CHCl3, 0.0013). IR (film, cm–1):
2960 (s), 1781 (s), 1729 (s), 1641 (w), 1462 (m), 1370 (s),
1244 (s), 1014 (s), 943 (s), 747 (m), 668 (m). HR-EI-MS
m/z (%): 226.1568 ([M+ – CH2CO], 100) (calcd. for
C13H22O3: 226.1569). EI-MS m/z (%): 226 (3.4), 225 (15.1),
208 (6.5), 207 (24.8), 168 (29.2), 165 (10.9), 150 (26.7), 138
(26.1), 110 (29.4), 99 (100), 81 (28.6), 71 (31.9).
The first fungal strain 5WS11I1 was isolated from a white
spruce (P. glauca (Moench voss)). This fungus was grown
on 2% malt extract agar (MEA) in 250 mL Erlenmeyer
flasks (2 L total) and Glaxo bottles (10 L total) according to
the established protocols (6). There were no other changes in
culturing procedures, and the fermentations proceeded si-
multaneously.
γ-Lactone 6
Isolation of compounds 1–11
An oil, [α]D = +105.8° (CHCl3, 0.0031). IR (film, cm–1):
3459 (br), 2962 (s), 1777 (s), 1713 (s), 1632 (m), 1420 (m),
1384 (m), 1213 (s), 1158 (s), 1030 (m). HR-EI-MS m/z (%):
168.0776 ([M+ – C4H8O], 5.0) (calcd. for C9H12O3:
168.0786), 167.0689 ([M+ – C4H9O], 18.1) (calcd. for
C9H11O3: 167.0708), 150 (31.0), 149 (20.1), 142 (13.0), 139
(73), 128 (23.6), 113 (16.1), 109 (30.4), 108 (26.9), 100
(19.9), 99 (100), 98 (12.1), 82 (91.6), 71 (82.7).
The culture filtrate, fermented in 250 mL Erlenmeyer
flasks, was extracted with EtOAc (3 × 600 mL). The crude
extract E (64.7 mg), obtained by removal of solvents under
vacuum at room temperature, was purified on Kieselgel 60
F254 plates (1 mm) using CHCl3–MeOH (9:1). The fraction
(23 mg, Rf = 0.2–0.8) was further purified by ptlc using
CHCl3–acetone (4:1) and yielded vermiculin 1 (1.7 mg)
(Kieselgel 60 F254, 0.5 mm, Rf = 0.70) and 7α,8β,11-tri-
hydroxydrimane 2 (3.4 mg) (Kieselgel 60 F254, 0.5 mm,
Rf = 0.11). The culture filtrate from the Glaxo bottles was
extracted with EtOAc (3 × 3 L), and the total extract was
evaporated to dryness at room temperature in vacuum to
yield crude extract G (0.972 g). A portion of the crude ex-
tracts (0.800 g) was chromatographed on a silica column
(Kieselgel 60G, 2.8 × 3.0 cm) and eluted with a discontinu-
ous gradient of CHCl3–MeOH (1:0–0:1). The fractions
(40 mL each) were combined on the basis of tlc scrutiny and
further purified by repeated ptlc, yielding 3 (1.5 mg) (Baker
Si-C18F, 0.20 mm, MeOH–H2O (4:1), Rf = 0.22), 4
(40.0 mg) (Baker Si-C18F, 0.20 mm, acetone–H2O (3:2),
Rf = 0.43), 5 (2.5 mg) (0.25 mm, hexane–EtOAc (3:2), Rf =
0.56), 6 (6.2 mg) (Baker Si-C18F, 0.20 mm, MeOH–H2O
(7:3), Rf = 0.62), 7 (6.3 mg) (Baker Si-C18F, 0.20 mm,
MeOH–H2O (13:7), Rf = 0.56), 8 (11.1 mg) (0.25 mm,
CHCl3–acetone (17:3), Rf = 0.62), 9 (5.7 mg) (Baker Si-
C18F, 0.20 mm, acetone–H2O (3:2), Rf = 0.69), and 10
(1.1 mg) (Baker Si-C18F, 0.20 mm, acetone–H2O (3:2), Rf =
0.54), as well as sequiterpenes 11 (12.0 mg), (0.25 mm,
CHCl3–MeOH (9:1), Rf = 0.47) and 7α,8β,11-trihydroxy-
drimane 2 (4.0 mg) (Baker Si-C18F, 0.20 mm, MeOH–H2O
(13:7), Rf = 0.25).
γ-Lactone 7
An oil, [α]D = +51.1° (CHCl3, 0.0032). IR (film, cm–1):
3430 (br), 2959 (s), 1775 (s), 1630 (w), 1458 (m), 1211 (s),
1014 (s), 942 (m), 749 (m). EI-MS m/z (%): 242 ([M+], 0.4),
224 ([M+ – H2O], 0.7), 206 ([M+ – 2H2O], 6.5), 171 (9.6),
170 (15.5), 152 (51.8), 110 (63.2), 99 (47.2), 83 (100), 82
(16.1), 71 (43).
γ-Lactone 8
An oil, [α]D = +45.6° (CHCl3, 0.0056). IR (film, cm–1):
3474 (br), 2960 (s), 1777 (s), 1711 (s), 1462 (m), 1368 (m),
1291 (m), 1214 (s), 1029 (s), 938 (s), 668 (s). HR-EI-MS
m/z (%): 241.1410 ([M+ – H], 6.8) (calcd. for C13H21O4:
241.1440), 226.1550 ([M+ + H – OH], 20.2) (calcd. for
C13H22O3: 226.1569), 225.1468 ([M+ – OH], 22.8) (calcd.
for C13H21O3: 225.1491), 224.1391 ([M+ – H2O], 9.9)
(calcd. for C13H20O3: 224.1412), 209 (23.5), 208 (12.2), 193
(16.3), 171 (16.4), 169 (31.5), 153 (22.2), 151 (30.5), 143
(30.7), 105 (28.9), 99 (79.4), 82 (51.8), 71 (38.4).
γ-Lactone 9
An oil, [α]D = +51.6° (CHCl3, 0.0029). IR (film, cm–1):
3417 (br), 2961 (s), 1777 (s), 1757 (s), 1454 (m), 1383 (m),
1217 (m), 1078 (s), 983 (m). EI-MS m/z (%): 257 ([M+ – H],
3.5) 256 ([M+ – 2H], 4.5), 240 ([M+ – H2O], 2.5), 186
([M+ – C4H8O], 6.5), 185 ([M+ – C4H9O], 3.5), 169 (18.5),
155 (15.0), 139 (26.5), 110 (45.0), 99 (49.50), 97 (64.6), 83
(65.6), 69 (98.2), 55 (100).
γ-Lactone 3
An oil, [α]D = +25.3° (CHCl3, 0.00075). IR (film, cm–1):
2959 (s), 1780 (s), 1640 (w), 1452 (m), 1382 (m), 1209 (s),
1157 (s), 940 (s), 748 (m). HR-EI-MS m/z (%): 210.1627
([M+], 100) (calcd. for C13H22O2: 210.1620). EI-MS m/z
(%): 210 (0.8), 209 (3.8), 166 (3.8), 149 (7.0), 99 (100), 96
(8.1), 81 (6.7), 71 (23.6).
γ-Lactone 10
An oil, [α]D = +65.5° (CHCl3, 0.0006). IR (film, cm–1):
3471 (br), 2958 (s), 1778 (s), 1723 (s), 1462 (m), 1382 (m),
1215 (s), 1158 (s), 1035 (s), 940 (m), 668 (m). EI-MS m/z
(%): 242 (2.8), 224 (4.9), 208 (14.6), 166 (10.2), 150 (25.9),
γ-Lactone 4
An oil, [α]D = +47.7° (CHCl3, 0.020). IR (film, cm–1):
3443 (br), 3020 (w), 2957 (s), 1777 (s), 1457 (s), 1210 (s),
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Findlay et al.
291
148 (13.1), 138 (10.6), 137 (13.1), 99 (100), 85 (22.4), 69
(41.6), 57 (29.4).
The culture was then macerated, and 50 mL was used to in-
oculate three Glaxo bottles containing CZ Met medium (1 L)
and three Glaxo bottles containing 2% malt extract medium
(1 L). These were incubated at 20°C for 6 weeks. After incu-
bation, each Glaxo bottle was filtered under vacuum though
Whatman #1 filter paper and extracted with three portions of
ethyl acetate (200 mL). The combined extract was dried
with anhydrous sodium sulfate, filtered, and concentrated
under vacuum at room temperature. The extracts were trans-
ferred into a pre-weighed vial using a small amount of ethyl
acetate, which was then evaporated under nitrogen. Com-
pounds 16–20 were variably isolated from a series of white
spruce and black spruce strains, but all were produced by
DAOM 229664 in CZ met medium incubated under the con-
ditions described. This isolate and DAOM 221611 have
been deposited in the Canadian Collection of Fungal Cul-
tures (DAOM, Agriculture Agri-food Canada, Ottawa, ON
K1A 0C6). They are preserved in liquid nitrogen.
Sesquiterpene 11
An oil, [α]D = +11.8° (CHCl3, 0.0039). IR (film, cm–1):
3391 (br, s), 2927 (s), 1694 (s), 1647 (s), 1381 (m), 1077
(m). HR-EI-MS m/z (%): 234.1636 ([M+ – 2H2O], 3.5)
(calcd. for C15H22O2: 234.1612), 211.1359 ([M+ – C3H7O],
19.5) (calcd. for C12H19O3: 211.1334), 206 (2.1), 194 (9.5),
193 (42.1), 163 (21.9), 143(58.1), 123 (28.9), 100 (40.3), 93
(33.5), 81 (76.2), 59 (100).
Synthesis of sesquiterpene 11
To a solution of 10-bromo-11-hydroxy-farnesylic acid 12
(0.220 g, 0.660 mmole) in DMF–H2O (3:1) (12.5 mL),
NaOH (0.055 g, 1.38 mmole) was added, and the solution
was stirred at room temperature for 1.5 h before quenching
with 0.01 M HCl. The mixture was extracted with CHCl3
(3 × 10.0 mL) and combined extracts washed with brine
(15 mL) and evaporated at room temperature under vacuum
to yield the crude epoxide 13 (0.197 g), which was dissolved
in DMF–H2O (3:1) (12.5 mL), and p-toluenesulfonic acid
(0.006 g, 0.003 mmole) was added. The solution was stirred
for 2 h before addition of brine (10.0 mL). The mixture was
extracted with CHCl3 (3 × 10.0 mL), and the combined ex-
tracts were washed with brine and evaporated under vacuum
to give a crude mixture, which was purified by column to
give sesquiterpene 11 (0.083 g, 46% from 12) and the
lactone 14 (0.060 g, 36% from 12).
Isolation of compounds 16–20
Endophyte crude extract 5WS19B2-AMSF OCT 22/98
(73.8 mg) was fractionated by ptlc (two plates) using
CHCl3–C6H12–CH3OH (7:2:1). Seven fractions A–G (in or-
der of increasing polarity) were collected. Fraction E
(20.8 mg, Rf 0.34 to 0.40) was purified by ptlc using C6H6–
CH3COCH3 (4:1) to afford pure compound 16 (0.65 mg, Rf
0.51) and pure compound 17 (7.1 mg, Rf 0.56). Fraction F
(21.2 mg, Rf 0.32) was purified by ptlc using C6H6–
CH3COCH3 to afford compound 19 (5.5 mg, Rf 0.20).
Endophyte crude extracts 5BS21B1-ACZGF, 5BS21B1-
BCZGF, and 5BS21B1-CCZGF NOV 30/98 were combined
(257.3 mg) and fractionated by ptlc (six plates) using
CHCl3–C6H12–CH3OH (7:2:1). Five fractions A–E (in order
of increasing polarity) were collected. Fraction D (58.1 mg)
was purified by ptlc with CH3COOC2H5–C6H12 (4:1) to give
two major fractions, one containing 18 (8.8 mg) and the
other a mixture of 19 and 16 (8.4 mg). Compound 18 was
further purified by ptlc with CH3COOC2H5–C6H12 (3:2),
(5.5 mg, Rf 0.52). Further purification of the mixture by ptlc
with CHCl3–C6H12–CH3OH (15:5:1) gave 19 (1.6 mg, Rf
0.38) and 16 (2.7 mg, Rf 0.42). Crude extract 5WS14G1-
ACZGF NOV 10/98 (34.7 mg) was fractionated by ptlc
(0.5 mm, two plates) using CHCl3–C6H12–CH3OH (7:2:1).
The major fraction (8.1 mg) was further purified by ptlc us-
ing CH3COOC2H5–C6H12 (4:1) to give pure compound 20
(3.1 mg, Rf 0.30)
Lactone 14
An oil. IR (film, cm–1): 3420 (s, br), 2972 (s), 1720 (s),
1
1640 (s), 1440 (s), 1376 (m), 1172 (s). H NMR (CDCl3,
ppm) δ: 5.69 (s), 5.08 (m), 4.83 (dd), 2.20 (m, 4H), 2.15 (s,
3H), 2.05–1.90 (m, 2H), 1.80–1.6 (m, 2H), 1.58 (s, 3H),
1.20 (s, 6H). 13C NMR (CDCl3, ppm) δ: 162.64 (C), 161.23
(C), 134.98(C), 123.89 (CH), 115.07 (CH), 79.56 (CH),
72.24(C), 40.92 (CH2), 35.88 (CH2), 27.64 (CH2), 26.52
(CH3), 25.76 (CH3), 25.76 (CH3), 24.85 (CH3), 19.09 (CH3),
15.93 (CH3). EI-MS m/z (%): 252 ([M+], 4.5), 211 (28.7),
193 (62.9), 163 (37.64), 143 (100), 135 (65.4), 125 (53.1),
100 (52.4).
Larval bioassays
Compounds 4, 6, 7, 8, 11, and 7α,8β,11-trihydroxydri-
mane 2 were subjected to a budworm larval feeding bioassay
according to established protocol (2) at a concentration of
0.0004 g g–1 of diet, resulting in mortalities of 60, 76, 68,
72, 52, and 72% relative to control (42% mortality).
6,7-Dihydroxy-2-propyl-2,4-octadien-4-olide 16
A pale yellow oil; [α]D = +17.1° (c 0.0021, MeOH). UV
(EtOH) λmax (nm) (ε): 272 (8600). HR-EI-MS m/z (%):
194.0962 ([M+
– H2O], 1.6) (calcd. for C11H14O3:
Fungal strain 5WS26H1 (DAOM 229664)
194.0943), 168 (100), 152 (11.6), 43 (29.8). See Table 3 for
spectral data.
Strains were transferred to slants containing 2% malt ex-
tract agar (Difco) and incubated at 20°C for at least one
month. For each fermentation, the slant was macerated in
sterile water (30 mL). An aliquot (2.5 mL) was used to inoc-
ulate each of six Erlenmeyer flasks (250 mL) containing
50 mL of medium (CZ Met) made from glucose (20 g),
NH4Cl (3 g), KH2PO4 (2 g), MgSO4 (2g), FeSO4·7H2O
(0.2 g), yeast extract (2 g), malt extract (2 g), and peptone
(2 g) per L of water. The flasks were put on a rotary shaker
(3.81 cm throw) at 220 rpm at 20°C for 48 h in the dark.
5,6,8-Trihydroxy-4-(1 -hydroxyethyl) isocoumarin 17
′
A yellow solid; mp 150–155°C; [α]D = –13.1° (c 0.007,
CH3COCH3). UV (EtOH) λmax (nm)(ε): 204 (8320), 238
(12 500), 252 (10 730), 272 (9820), 348 (9120). HR-EI-MS
m/z (%): 238.0467 ([M+], 10.8) (calcd. for C11H10O6:
238.0477), 220 ([M+ – H2O], 100), 205 (36.9), 43 (16). See
Table 4 for spectral data.
© 2003 NRC Canada

292
Can. J. Chem. Vol. 81, 2003
CF1 was a gift from Guido Caputo, Canadian Forestry Ser-
vice, Sault Ste. Marie, Ontario, Canada. Thanks are also ow-
ing to Ghislain Deslongchamps for the Merck MMF 94
conformational search and to John A. Johnson for providing
the larval bioassays.
4-Hydroxy-2-methoxyacetanilide 20
A yellow amorphous solid; mp 166–169°C (synthetic
1
(synth.) 169–171°C). H NMR (CD3OD, 400 MHz) δ: 8.57,
7.53 (1H, br s, 4-OH and 1H, s, NH measured in CDCl3 + 5
drops of DMSO-d6), 7.48 (1H, d, J = 8.0 Hz, H-6), 6.45
(1H, d, J = 2.4, 9.6, H-5), 3.81 (1H, s, OCH3), 2.1 (1H, s,
CH3). 13C NMR (CD3OD, MHz) δ: 170.4 (C=O), 155.6 (C-
4), 152.4 (C-2), 124.5 (C-6), 118.3 (C-1), 105.9 (C-5), 98.7
(C-3), 54.6 (OCH3), 21.9 (CH3). HR-EI-MS m/z (%):
181.0740 ([M+], 60) (calcd. for C9H11NO3: 181.0739), 139
(70), 124 (100).
References
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Cell line assays
The spruce budworm cell line CF-1 was used according to
the method of Clark et al. (9) to screen extracts and purified
compounds for cytotoxicity. Deoyxnivalenol (2 µg mL–1, cell
culture medium) was used as the positive control and etha-
nol (2 µL mL–1) as the negative control; no effect of ethanol
on the CF-1 cells was detected at this concentration. Crude
extracts were dissolved in ethanol and tested at 1, 2, and
4 µL mL–1 culture medium. The crude extract arising from
the non-sporulating strain 5WS26H1 (DAOM 229664) was
as cytotoxic as the positive control solution. Compounds 16–
19 were individually dissolved in ethanol and tested at
2 µg mL–1 culture medium as above. Compound 17, sescan-
delin 18, and sescandelin B 19 were individually weakly
cytotoxic. Compound 16 was ca. 20–30% as cytotoxic to
CF-1 as deoxynivalenol at 2 µL mL–1 culture medium.
12. S. Tahara, K. Fujiwara, and J. Mizutzni. Agric. Biol. Chem.
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Acknowledgments
Thanks are owing to the Natural Sciences and Engi-
neering Research Council of Canada (NSERC), the Forestry
Canada Green Plan (Research Network for the Discovery
and Development of Natural Products for Integrated Forest
Pest Management), the Industrial Research Assistance Pro-
gram (IRAP) of the National Research Council of Canada
(NRC), and JD Irving Ltd. Technical assistance was pro-
vided by Don Belilse, Kristina Bramwell, Regina de la
Campa, Mark Foto, and Sabrena MacKenzie. The cell line
17. H. Raistrick and C.E. Stickings. Biochem. J. 48, 53 (1951).
18. V.C. Vora. J. Sci. Ind. Res. 13b, 842 (1954).
© 2003 NRC Canada